Wnt5a基因在乳腺癌中表达和临床意义及其机制的研究
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摘要
目的
在临床上激素受体阳性的乳腺癌患者的预后较阴性的患者好,激素受体阳性也是运用内分泌治疗的指征,因此调节激素受体的表达具有重要的临床意义。miRNA-206通过与雌激素受体(ER)3’-非翻译区结合从而抑制ER的表达。术后辅助内分泌治疗联合唑来膦酸研究结果显示其可能具有直接的抗肿瘤作用以及增强内分泌治疗疗效的作用。Wnt信号通路和肿瘤的发生发展密切相关,其中Wnt5a的表达与乳腺癌激素受体表达密切相关,但是Wnt5a基因对乳腺癌生物学行为影响、调控雌激素受体表达的机制以及唑来膦酸增强内分泌疗效的作用机制尚无深入研究。本课题旨在研究Wnt5a基因对人乳腺癌细胞生物学行为的影响和在乳腺癌中表达的临床意义以及此基因多态性与乳腺癌发病风险以及激素受体表达间的相关性,探索其调节雌激素受体表达可能的机制。
方法
第一部分
1.设计并合成对人Wnt5a基因的RNA干扰片段,将siRNA片段瞬转入ER阳性人乳腺癌细胞株下调其Wnt5a基因表达水平,用Western-blot方法验证下调Wnt5a蛋白表达的有效性,观察下调此基因后对细胞ER蛋白表达的影响；分别用CCK-8试剂盒以及Transwell小室测定Wnt5a基因下调后对细胞增殖水平以及体外侵袭能力的影响。
2.用实时定量PCR(real-time PCR)检测ER阳性和ER阴性人乳腺癌细胞株miRNA-206表达差异以及下调ER阳性人乳腺癌细胞株Wnt5a基因表达水平后对miRNA-206表达的影响。
3.用mimic-miRNA-206和inhibitor-miRNA-206瞬时转染ER阳性人乳腺癌细胞株,用real-time PCR验证上调和下调miRNA-206表达水平的有效性并观察此小RNA表达水平的改变对Wnt5a以及ER蛋白表达的影响。
4.采用CCK-8试剂盒以及PI染色流式细胞计数法(flow cytometry, FCM)检测唑来膦酸对ER阳性人乳腺癌细胞增殖以及凋亡的影响。
5. Western-blot法检测唑来膦酸对ER阳性人乳腺癌细胞株ER、Wnt5a以及自噬蛋白表达水平的影响。
6.CCK-8试剂盒检测下调ER阳性人乳腺癌细胞Wnt5a基因后对唑来膦酸敏感性的影响
7.透射电镜观察唑来膦酸诱导乳腺癌细胞产生自噬体。
8.用CCK-8试剂盒检测唑来膦酸联合氟维斯群对人乳腺癌细胞株增殖能力的影响,Western-blot法检测两药联合对乳腺癌细胞ER、Wnt5a蛋白表达的影响。
第二部分
1.收集乳腺癌患者741例以及对照良性乳腺疾病患者293例,病例组与对照组根据年龄匹配,签署知情同意书后获取外周血5ml。
2.提取外周血DNA,对于Wnt5a的单核苷酸多态性(SNP)位点进行检测。候选SNP位点由ABI公司开发的SNPbrowserTM v4.0软件来选择。
3.对于Wnt5a基因多态性位点与乳腺癌易感性以及乳腺癌患者中Wnt5a基因多态性与临床病理特征之间的关系进行统计学分析。
第三部分
收集2005年01月～2005年12月间复旦大学附属肿瘤医院病理确诊的女性原发性乳腺癌标本218例临床病理资料和肿瘤组织蜡块标本,对所有入组病例进行随访,运用免疫组化方法检测乳腺癌肿瘤组织中Wnt5a的表达。研究Wnt5a的表达与乳腺癌患者临床病理特征以及预后的关系。分析其表达与临床病理特征以及预后的关系采用卡方检验。生存曲线用Kaplan-Meier法计算。多因素分析采用cox比例风险模型进行,以P<0.05为差异有统计学意义。数据分析采用stata10.0软件进行统计学分析。
结果
第一部分
1.下调MCF-7细胞Wnt5a基因表达水平后可以抑制ER蛋白的表达,并且可以促进细胞增殖,但对细胞侵袭能力无影响。
2.ER阳性的乳腺癌细胞株MCF-7的miRNA-206表达水平低于ER阴性的乳腺癌细胞株MDA-MB-231细胞株；下调MCF-7细胞Wnt5a基因表达后miRNA-206表达增高。
3.上调MCF-7细胞miRNA-206表达后可抑制其Wnt5a以及ER蛋白的表达水平；抑制MCF-7细胞miRNA-206表达后对Wnt5a以及ER蛋白表达无明显影响。
4.唑来膦酸可以抑制MCF-7细胞增殖、诱导其凋亡并且具有浓度依赖性。
5.唑来膦酸可以抑制MCF-7细胞株的Wnt5a以及ER蛋白的表达,另外还可以诱导自噬标记蛋白LC3-Ⅱ表达增多。
6.下调MCF-7细胞株的Wnt5a基因表达水平后对唑来膦酸的敏感性降低。
7.透射电镜下可以观察到唑来膦酸诱导MCF-7产生自噬体,并且随着浓度的增高自噬体形成增多。
8.唑来膦酸和氟维斯群两药合用比唑来膦酸和氟维斯群单药对MCF-7细胞株抑制作用更好；两药合用可以明显降低MCF-7细胞Wnt5a以及ER蛋白的表达。
第二部分
1.校正了年龄、绝经状态和乳腺癌家族史三个因素后多因素logistic回归分析结果发现Wnt5a的rs10865994、rs11918967和rs472631这三个SNP位点均与乳腺癌发病风险无关。
2.在乳腺癌患者中rs11918967这个SNP位点在孕激素受体(PR)阳性和阴性组分布频率差异有统计学意义(P<0.05)。杂合(CG)及纯合变异基因型(CC)相对于纯和野生基因型(GG)可增加孕激素受体阳性率以及激素受体阳性率(激素受体阳性指ER和PR阳性)
3.rs11918967位点CG/GG基因型在肿瘤大小,腋下淋巴结转移状态、HR/ERBB2表型两组间无明显差异。
第三部分
1.在218例乳腺癌患者中,117例患者Wnt5a阴性,101例患者Wnt5a阳性,阳性率为46.33%。
2.Wnt5a阳性的患者的肿瘤明显小于阴性的患者(P=0.02)。Wnt5a的表达和ERa,PR的表达有明显的相关性,Wnt5a阳性的患者中ERa,PR的阳性率高(P=0.046和P=0.017)。与Wnt5a阳性组相比,Wnt5a阴性的病人更容易复发转移(P=0.023)。
3.单因素生存分析发现Wnt5a表达阳性的患者无复发生存率比Wnt5a阴性的患者低(P=0.026),Wnt5a阳性组5年生存率为89.35%,Wnt5a阴性组为72.60%。原发肿瘤大于2cm的患者预后差于小于等于2cm的患者(P=0.017),淋巴结阳性的患者预后比淋巴结阴性的患者预后差(P=0.027)。亚组分析中发现,在ERα阳性患者中Wnt5a的阳性表达的预后优于阴性表达的患者(P=0.035而在ERα阴性的患者中是否表达Wnt5a与预后不相关(P=0.128)。
4.采用多因素Cox比例风险模型校正后,Wnt5a(P=0.028,RR=0.38,95%C10.16-0.090)和淋巴结转移情况(P=0.039,RR=2.36,95%CI1.04-5.33)均是无复发生存率的独立预后因素。在ERα阳性患者中,多因素分析结果也提示了Wnt5a在ERα阳性的乳腺癌患者中也是一个独立的预测预后的因素。
5.从复发风险图可以看出Wnt5a阴性的患者复发峰值出现在39个月,Wnt5a阳性的患者的复发风险明显低于Wnt5a阴性组患者,其组最早的峰值出现的也比阴性组患者晚,在术后60个月之后。
结论
1.Wnt5a在乳腺癌中是一个抑癌基因,可能与miRNA-206相互作用共同调节ERα的表达。唑来膦酸具有独立的抗肿瘤作用并且可以增强内分泌治疗的疗效,这种增效作用的机制可能部分通过调节Wnt5a-ERα这条通路来实现的。
2.Wnt5a基因多态性与乳腺癌患者激素受体表达密切相关,这将为以后建立一个基于基因数据的乳腺癌内分泌治疗效果的预测以及预后预测模型奠定良好的基础。
3.在乳腺癌中Wnt5a的表达与ERα,PR的表达密切相关,Wnt5a阳性的患者的无复发生存率优于阴性的患者,是一个独立的预测乳腺癌患者预后的指标。
Objective
Hormone receptor positive breast cancer patients had better prognosis than negative cancers, hormone receptor positive was the indication of endocrine therapy. Therefore, regulation of hormone receptor expression has important clincical significance. Wnt signal pathway was closely correlated to the genesis of tumors, Wnt5a was significantly correclated with the status of hormone receptor. But the effect of Wnt5a on breast cancer biological characteristics and the mechanisms of regulation hormone receptor expression need to be further study. This thesis amis at investigating the effects of Wnt5a on biological characteristics of breast cancer cells, clinical significance of Wnt5a in the patients with breast cancer and the association between polymorphisms of Wnt5a and the occur and the hormone receptor status of breast cancer.
Methods
Part I
1. SiRNA for Wnt5a gene was designed and synthesized, then be transfeted into breast cancer cells line MCF-7. Western-blot was used to screen the most effective siRNA and Wnt5a protein changes. The effects of Wnt5a gene downregulation on proliferation and invasive ability were tested by CCK-8and transwell.
2. The difference of miRNA-206expression between MCF-7and MDA-MB-231cell lines was determined by real-time quantitative PCR. Downregulation of MCF-7Wnt5a mRNAthen detected the miRNA-206changes by real-time PCR.
3. Mimic-miRNA-206and inhibitor-miRNA-206were transfected into ER-positive breast cancer cell lines, transfect effiency was assessed by real-time PCR. Expression of Wnt5a and ER protein was detected by western-blot.
4. Effects of zoledronic acid on the proliferation, apoptosis in ER-positive breast cancer cells were investigated by CCK-8and FCM.
5. CCK-8was used to observe the effect of Wnt5a downregulation on zoledronic acid sensitivity.
6. The protein expression of Wnt5a, ER and LC3in different concentration zoledronic acid treated ER-positive breast cancer cells were detected by western-blot.
7. Autophagy indued by zoledronic acid was investigated by transmission electron microscope.
8. CCK-8was used to detect the effects of zoledronic acid combined with fulvestrant on growth of human breast cancer cell line MCF-7, western-blot was employed to detect the expression of Wnt5a and ER of human breast caner cells treated by the combination of zoledronic acid and fulvestrant.
Part Ⅱ
1. We used741breast cancer cases and293cancer free controls, controls were matched by age. Took5.0ml venous blood from every recruited patients after signing consent.
2. To extract the DNA and to determine the association between SNPs in genes encoding Wnt5a and breast cancer risk. SNP genotyping was used MassARRAY system by means of matrix assited laser desorption ionization-time of flight mass spectrometry method (MALEI-TOF) according to the manufacturers instructions.
3. We evaluated the odds ratios and their95%confident intervals of the associations between Wnt5a polymorphism and breast cancer risk and hormone receptor status among breast cancer patients.
Part Ⅲ
218patients of primary breast cancer resected between January2005to December2005in Fudan University Shanghai Cancer Center were included. Clinicopathologic data were obtained from patients" records. Immunohistochemical method was used to detect the expression of Wnt5a. The correlation among Wnt5a and clinicopathologic factors and survival were then statistically analyzed, using stata10.0. The statistical associations between clinical parameters and immunohistochemical staining were tested by y2test. The survival rate was calculated by the Kaplan-Meier method. Variables that seemed to be significantly associated with survival on univariate analysis were entered into multivariate analysis, which was performed with Cox proportional hazard model. A P value<0.05was considered significant.
Results
Part Ⅰ
1. Downregulation MCF-7cell Wnt5a mRNA could inhibit the expression of ER protein and promote MCF-7cell proliferation, but had no effect on the invation of MCF-7cells.
2. The expression level of miRNA-206in MCF-7cells was lower than MDA-MB-231cells. Downregulation of MCF-7cells Wnt5a mRAN could enhance the expression of miRNA-206.
3. Upregulation miRNA-206of MCF-7cells could inhibit the protein expression of Wnt5a and ER, downregulation miRNA-206of MCF-7cells had no effect on the expression of Wnt5a and ER protein.
4. Zoledronic acid could significantly decreased proliferation and induced apoptosis in breast cancer MCF-7cell line which depended on drug concentration.
5. Human breast cancer MCF-7cells transfected by siRNA-Wnt5a could obviously decreased the sensitivity of MCF-7cells to zoledronic acid.
6. Zoledronic acid could significanctly decreased the protein expression of Wnt5a and ER in MCF-7cells, and enhanced LC3protein expression.
7. Autophagosomes in MCF-7cells induced by zoledronic acid were observed by transmission electron microscopy, and autophagosome formation enhanced with concentrations increased.
8. Combination treatment of zoledronic acid and fulvestrant could significantly inhibit the growth of MCF-7cells, the protein expression of Wnt5a and ER decreased in MCF-7cells exposed to the combination of the two drugs.
Part Ⅱ
Multiple factor logistic regression analysis demonstrated that the polymorphism of Wnt5a was not associated with breast cancer risk after controlling the age, meno status and breast cancer family history.In the group of breast cancer, we found CG+CC genotype compared to GG genotype of SNP rsl1918967increased the positive proportion of PR and HR.
Part Ⅲ
High expression rates of Wnt5a was46.33%. A statistically significant association between the expression of Wnt5a correlated with tumor size, ER and PR. In the univariate survival analysis, we observed Wnt5a negative patients were more likey to recur than those Wnt5a positive patients. The Cox proportion hazards regression analysis showed that Wnt5a expression as well as lymph nodes was an independent prognostic factor for relapse-free survival. In subgroup of ER positive breast cancer patients, Wnt5a was also a independent prognostic factor.
Conclusion
1. Wnt5a gene is an important tumor suppressor gene in breast cancer, it might act together with miRNA-206to control the expression of ER. Zoledronic acid had a direct anti-tumor effect and enhanced the action of endocrine therapy, and this might be achieved through regulating Wnt5a-ER pathway.
2. The polymorphism of Wnt5a gene have an effect on the ER and HR expression in breast cancer patients. It will lay the foundation of predicting endocrine therapy effect and prognosis model based on gene data.
3. Expression of Wnt5a was significantly correlated with ER and PR. The survival rate of Wnt5a positive breast cancer patients was superior to negative patients, it was an independent prognostic factor.
在临床上激素受体阳性的乳腺癌患者的预后较阴性的患者好,激素受体阳性也是运用内分泌治疗的指征,因此调节激素受体的表达具有重要的临床意义。miRNA-206通过与雌激素受体(ER)3’-非翻译区结合从而抑制ER的表达。术后辅助内分泌治疗联合唑来膦酸研究结果显示其可能具有直接的抗肿瘤作用以及增强内分泌治疗疗效的作用。Wnt信号通路和肿瘤的发生发展密切相关,其中Wnt5a的表达与乳腺癌激素受体表达密切相关,但是Wnt5a基因对乳腺癌生物学行为影响、调控雌激素受体表达的机制以及唑来膦酸增强内分泌疗效的作用机制尚无深入研究。本课题旨在研究Wnt5a基因对人乳腺癌细胞生物学行为的影响和在乳腺癌中表达的临床意义以及此基因多态性与乳腺癌发病风险以及激素受体表达间的相关性,探索其调节雌激素受体表达可能的机制。
方法
第一部分
1.设计并合成对人Wnt5a基因的RNA干扰片段,将siRNA片段瞬转入ER阳性人乳腺癌细胞株下调其Wnt5a基因表达水平,用Western-blot方法验证下调Wnt5a蛋白表达的有效性,观察下调此基因后对细胞ER蛋白表达的影响；分别用CCK-8试剂盒以及Transwell小室测定Wnt5a基因下调后对细胞增殖水平以及体外侵袭能力的影响。
2.用实时定量PCR(real-time PCR)检测ER阳性和ER阴性人乳腺癌细胞株miRNA-206表达差异以及下调ER阳性人乳腺癌细胞株Wnt5a基因表达水平后对miRNA-206表达的影响。
3.用mimic-miRNA-206和inhibitor-miRNA-206瞬时转染ER阳性人乳腺癌细胞株,用real-time PCR验证上调和下调miRNA-206表达水平的有效性并观察此小RNA表达水平的改变对Wnt5a以及ER蛋白表达的影响。
4.采用CCK-8试剂盒以及PI染色流式细胞计数法(flow cytometry, FCM)检测唑来膦酸对ER阳性人乳腺癌细胞增殖以及凋亡的影响。
5. Western-blot法检测唑来膦酸对ER阳性人乳腺癌细胞株ER、Wnt5a以及自噬蛋白表达水平的影响。
6.CCK-8试剂盒检测下调ER阳性人乳腺癌细胞Wnt5a基因后对唑来膦酸敏感性的影响
7.透射电镜观察唑来膦酸诱导乳腺癌细胞产生自噬体。
8.用CCK-8试剂盒检测唑来膦酸联合氟维斯群对人乳腺癌细胞株增殖能力的影响,Western-blot法检测两药联合对乳腺癌细胞ER、Wnt5a蛋白表达的影响。
第二部分
1.收集乳腺癌患者741例以及对照良性乳腺疾病患者293例,病例组与对照组根据年龄匹配,签署知情同意书后获取外周血5ml。
2.提取外周血DNA,对于Wnt5a的单核苷酸多态性(SNP)位点进行检测。候选SNP位点由ABI公司开发的SNPbrowserTM v4.0软件来选择。
3.对于Wnt5a基因多态性位点与乳腺癌易感性以及乳腺癌患者中Wnt5a基因多态性与临床病理特征之间的关系进行统计学分析。
第三部分
收集2005年01月～2005年12月间复旦大学附属肿瘤医院病理确诊的女性原发性乳腺癌标本218例临床病理资料和肿瘤组织蜡块标本,对所有入组病例进行随访,运用免疫组化方法检测乳腺癌肿瘤组织中Wnt5a的表达。研究Wnt5a的表达与乳腺癌患者临床病理特征以及预后的关系。分析其表达与临床病理特征以及预后的关系采用卡方检验。生存曲线用Kaplan-Meier法计算。多因素分析采用cox比例风险模型进行,以P<0.05为差异有统计学意义。数据分析采用stata10.0软件进行统计学分析。
结果
第一部分
1.下调MCF-7细胞Wnt5a基因表达水平后可以抑制ER蛋白的表达,并且可以促进细胞增殖,但对细胞侵袭能力无影响。
2.ER阳性的乳腺癌细胞株MCF-7的miRNA-206表达水平低于ER阴性的乳腺癌细胞株MDA-MB-231细胞株；下调MCF-7细胞Wnt5a基因表达后miRNA-206表达增高。
3.上调MCF-7细胞miRNA-206表达后可抑制其Wnt5a以及ER蛋白的表达水平；抑制MCF-7细胞miRNA-206表达后对Wnt5a以及ER蛋白表达无明显影响。
4.唑来膦酸可以抑制MCF-7细胞增殖、诱导其凋亡并且具有浓度依赖性。
5.唑来膦酸可以抑制MCF-7细胞株的Wnt5a以及ER蛋白的表达,另外还可以诱导自噬标记蛋白LC3-Ⅱ表达增多。
6.下调MCF-7细胞株的Wnt5a基因表达水平后对唑来膦酸的敏感性降低。
7.透射电镜下可以观察到唑来膦酸诱导MCF-7产生自噬体,并且随着浓度的增高自噬体形成增多。
8.唑来膦酸和氟维斯群两药合用比唑来膦酸和氟维斯群单药对MCF-7细胞株抑制作用更好；两药合用可以明显降低MCF-7细胞Wnt5a以及ER蛋白的表达。
第二部分
1.校正了年龄、绝经状态和乳腺癌家族史三个因素后多因素logistic回归分析结果发现Wnt5a的rs10865994、rs11918967和rs472631这三个SNP位点均与乳腺癌发病风险无关。
2.在乳腺癌患者中rs11918967这个SNP位点在孕激素受体(PR)阳性和阴性组分布频率差异有统计学意义(P<0.05)。杂合(CG)及纯合变异基因型(CC)相对于纯和野生基因型(GG)可增加孕激素受体阳性率以及激素受体阳性率(激素受体阳性指ER和PR阳性)
3.rs11918967位点CG/GG基因型在肿瘤大小,腋下淋巴结转移状态、HR/ERBB2表型两组间无明显差异。
第三部分
1.在218例乳腺癌患者中,117例患者Wnt5a阴性,101例患者Wnt5a阳性,阳性率为46.33%。
2.Wnt5a阳性的患者的肿瘤明显小于阴性的患者(P=0.02)。Wnt5a的表达和ERa,PR的表达有明显的相关性,Wnt5a阳性的患者中ERa,PR的阳性率高(P=0.046和P=0.017)。与Wnt5a阳性组相比,Wnt5a阴性的病人更容易复发转移(P=0.023)。
3.单因素生存分析发现Wnt5a表达阳性的患者无复发生存率比Wnt5a阴性的患者低(P=0.026),Wnt5a阳性组5年生存率为89.35%,Wnt5a阴性组为72.60%。原发肿瘤大于2cm的患者预后差于小于等于2cm的患者(P=0.017),淋巴结阳性的患者预后比淋巴结阴性的患者预后差(P=0.027)。亚组分析中发现,在ERα阳性患者中Wnt5a的阳性表达的预后优于阴性表达的患者(P=0.035而在ERα阴性的患者中是否表达Wnt5a与预后不相关(P=0.128)。
4.采用多因素Cox比例风险模型校正后,Wnt5a(P=0.028,RR=0.38,95%C10.16-0.090)和淋巴结转移情况(P=0.039,RR=2.36,95%CI1.04-5.33)均是无复发生存率的独立预后因素。在ERα阳性患者中,多因素分析结果也提示了Wnt5a在ERα阳性的乳腺癌患者中也是一个独立的预测预后的因素。
5.从复发风险图可以看出Wnt5a阴性的患者复发峰值出现在39个月,Wnt5a阳性的患者的复发风险明显低于Wnt5a阴性组患者,其组最早的峰值出现的也比阴性组患者晚,在术后60个月之后。
结论
1.Wnt5a在乳腺癌中是一个抑癌基因,可能与miRNA-206相互作用共同调节ERα的表达。唑来膦酸具有独立的抗肿瘤作用并且可以增强内分泌治疗的疗效,这种增效作用的机制可能部分通过调节Wnt5a-ERα这条通路来实现的。
2.Wnt5a基因多态性与乳腺癌患者激素受体表达密切相关,这将为以后建立一个基于基因数据的乳腺癌内分泌治疗效果的预测以及预后预测模型奠定良好的基础。
3.在乳腺癌中Wnt5a的表达与ERα,PR的表达密切相关,Wnt5a阳性的患者的无复发生存率优于阴性的患者,是一个独立的预测乳腺癌患者预后的指标。
Objective
Hormone receptor positive breast cancer patients had better prognosis than negative cancers, hormone receptor positive was the indication of endocrine therapy. Therefore, regulation of hormone receptor expression has important clincical significance. Wnt signal pathway was closely correlated to the genesis of tumors, Wnt5a was significantly correclated with the status of hormone receptor. But the effect of Wnt5a on breast cancer biological characteristics and the mechanisms of regulation hormone receptor expression need to be further study. This thesis amis at investigating the effects of Wnt5a on biological characteristics of breast cancer cells, clinical significance of Wnt5a in the patients with breast cancer and the association between polymorphisms of Wnt5a and the occur and the hormone receptor status of breast cancer.
Methods
Part I
1. SiRNA for Wnt5a gene was designed and synthesized, then be transfeted into breast cancer cells line MCF-7. Western-blot was used to screen the most effective siRNA and Wnt5a protein changes. The effects of Wnt5a gene downregulation on proliferation and invasive ability were tested by CCK-8and transwell.
2. The difference of miRNA-206expression between MCF-7and MDA-MB-231cell lines was determined by real-time quantitative PCR. Downregulation of MCF-7Wnt5a mRNAthen detected the miRNA-206changes by real-time PCR.
3. Mimic-miRNA-206and inhibitor-miRNA-206were transfected into ER-positive breast cancer cell lines, transfect effiency was assessed by real-time PCR. Expression of Wnt5a and ER protein was detected by western-blot.
4. Effects of zoledronic acid on the proliferation, apoptosis in ER-positive breast cancer cells were investigated by CCK-8and FCM.
5. CCK-8was used to observe the effect of Wnt5a downregulation on zoledronic acid sensitivity.
6. The protein expression of Wnt5a, ER and LC3in different concentration zoledronic acid treated ER-positive breast cancer cells were detected by western-blot.
7. Autophagy indued by zoledronic acid was investigated by transmission electron microscope.
8. CCK-8was used to detect the effects of zoledronic acid combined with fulvestrant on growth of human breast cancer cell line MCF-7, western-blot was employed to detect the expression of Wnt5a and ER of human breast caner cells treated by the combination of zoledronic acid and fulvestrant.
Part Ⅱ
1. We used741breast cancer cases and293cancer free controls, controls were matched by age. Took5.0ml venous blood from every recruited patients after signing consent.
2. To extract the DNA and to determine the association between SNPs in genes encoding Wnt5a and breast cancer risk. SNP genotyping was used MassARRAY system by means of matrix assited laser desorption ionization-time of flight mass spectrometry method (MALEI-TOF) according to the manufacturers instructions.
3. We evaluated the odds ratios and their95%confident intervals of the associations between Wnt5a polymorphism and breast cancer risk and hormone receptor status among breast cancer patients.
Part Ⅲ
218patients of primary breast cancer resected between January2005to December2005in Fudan University Shanghai Cancer Center were included. Clinicopathologic data were obtained from patients" records. Immunohistochemical method was used to detect the expression of Wnt5a. The correlation among Wnt5a and clinicopathologic factors and survival were then statistically analyzed, using stata10.0. The statistical associations between clinical parameters and immunohistochemical staining were tested by y2test. The survival rate was calculated by the Kaplan-Meier method. Variables that seemed to be significantly associated with survival on univariate analysis were entered into multivariate analysis, which was performed with Cox proportional hazard model. A P value<0.05was considered significant.
Results
Part Ⅰ
1. Downregulation MCF-7cell Wnt5a mRNA could inhibit the expression of ER protein and promote MCF-7cell proliferation, but had no effect on the invation of MCF-7cells.
2. The expression level of miRNA-206in MCF-7cells was lower than MDA-MB-231cells. Downregulation of MCF-7cells Wnt5a mRAN could enhance the expression of miRNA-206.
3. Upregulation miRNA-206of MCF-7cells could inhibit the protein expression of Wnt5a and ER, downregulation miRNA-206of MCF-7cells had no effect on the expression of Wnt5a and ER protein.
4. Zoledronic acid could significantly decreased proliferation and induced apoptosis in breast cancer MCF-7cell line which depended on drug concentration.
5. Human breast cancer MCF-7cells transfected by siRNA-Wnt5a could obviously decreased the sensitivity of MCF-7cells to zoledronic acid.
6. Zoledronic acid could significanctly decreased the protein expression of Wnt5a and ER in MCF-7cells, and enhanced LC3protein expression.
7. Autophagosomes in MCF-7cells induced by zoledronic acid were observed by transmission electron microscopy, and autophagosome formation enhanced with concentrations increased.
8. Combination treatment of zoledronic acid and fulvestrant could significantly inhibit the growth of MCF-7cells, the protein expression of Wnt5a and ER decreased in MCF-7cells exposed to the combination of the two drugs.
Part Ⅱ
Multiple factor logistic regression analysis demonstrated that the polymorphism of Wnt5a was not associated with breast cancer risk after controlling the age, meno status and breast cancer family history.In the group of breast cancer, we found CG+CC genotype compared to GG genotype of SNP rsl1918967increased the positive proportion of PR and HR.
Part Ⅲ
High expression rates of Wnt5a was46.33%. A statistically significant association between the expression of Wnt5a correlated with tumor size, ER and PR. In the univariate survival analysis, we observed Wnt5a negative patients were more likey to recur than those Wnt5a positive patients. The Cox proportion hazards regression analysis showed that Wnt5a expression as well as lymph nodes was an independent prognostic factor for relapse-free survival. In subgroup of ER positive breast cancer patients, Wnt5a was also a independent prognostic factor.
Conclusion
1. Wnt5a gene is an important tumor suppressor gene in breast cancer, it might act together with miRNA-206to control the expression of ER. Zoledronic acid had a direct anti-tumor effect and enhanced the action of endocrine therapy, and this might be achieved through regulating Wnt5a-ER pathway.
2. The polymorphism of Wnt5a gene have an effect on the ER and HR expression in breast cancer patients. It will lay the foundation of predicting endocrine therapy effect and prognosis model based on gene data.
3. Expression of Wnt5a was significantly correlated with ER and PR. The survival rate of Wnt5a positive breast cancer patients was superior to negative patients, it was an independent prognostic factor.
引文
1. Siegel R, Naishadham D, and Jemal A, Cancer statistics,2012[J]. CA Cancer J Clin,2012.62(1):10-29.
2. Anderson WF, Chatterjee N, Ershler WB, et al., Estrogen receptor breast cancer phenotypes in the Surveillance, Epidemiology, and End Results database[J]. Breast Cancer Res Treat,2002.76(1):27-36.
3. Murphy LC and Watson P, Steroid receptors in human breast tumorigenesis and breast cancer progression[J]. Biomed Pharmacother,2002.56(2):65-77.
4. Dunnwald LK, Rossing MA, and Li CI, Hormone receptor status, tumor characteristics, and prognosis:a prospective cohort of breast cancer patients[J]. Breast Cancer Res,2007.9(1):R6.
5. Johnston SR, Acquired tamoxifen resistance in human breast cancer--potential mechanisms and clinical implications[J]. Anticancer Drugs,1997.8(10): 911-30.
6. Logan CY and Nusse R, The Wnt signaling pathway in development and disease[J]. Annu Rev Cell Dev Biol,2004.20:781-810.
7. Peifer M and Polakis P, Wnt signaling in oncogenesis and embryogenesis--a look outside the nucleus[J]. Science,2000.287(5458):1606-9.
8. Kuhl M, Sheldahl LC, Park M, et al., The Wnt/Ca2+pathway:a new vertebrate Wnt signaling pathway takes shape [J]. Trends Genet,2000.16(7): 279-83.
9. Howng SL, Wu CH, Cheng TS, et al., Differential expression of Wnt genes, beta-catenin and E-cadherin in human brain tumors[J]. Cancer Lett,2002. 183(1):95-101.
10. Weeraratna AT, Jiang Y, Hostetter G, et al., Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma[J]. Cancer Cell,2002.1(3): 279-88.
11. Kurayoshi M, Oue N, Yamamoto H, et al., Expression of Wnt-5a is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion[J]. Cancer Res,2006.66(21):10439-48.
12. Ripka S, Konig A, Buchholz M, et al., WNT5A--target of CUTL1 and potent modulator of tumor cell migration and invasion in pancreatic cancer [J]. Carcinogenesis,2007.28(6):1178-87.
13. Liang H, Chen Q, Coles AH, et al., Wnt5a inhibits B cell proliferation and functions as a tumor suppressor in hematopoietic tissue[J]. Cancer Cell,2003. 4(5):349-60.
14. Dejmek J, Dejmek A, Safholm A, et al., Wnt-5a protein expression in primary dukes B colon cancers identifies a subgroup of patients with good prognosis [J]. Cancer Res,2005.65(20):9142-6.
15. Jonsson M, Dejmek J, Bendahl PO, et al., Loss of Wnt-5a protein is associated with early relapse in invasive ductal breast carcinomas[J]. Cancer Res,2002. 62(2):409-16.
16. Ford CE, Ekstrom EJ, and Andersson T, Wnt-5a signaling restores tamoxifen sensitivity in estrogen receptor-negative breast cancer cells[J]. Proc Natl Acad Sci USA,2009.106(10):3919-24.
17. Bartel DP, MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell,2004.116(2):281-97.
18. Lee RC, Feinbaum RL, and Ambros V, The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell, 1993.75(5):843-54.
19. Wightman B, Ha I, and Ruvkun G, Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans[J]. Cell,1993.75(5):855-62.
20. Doench JG, Petersen CP, and Sharp PA, siRNAs can function as miRNAs[J]. Genes Dev,2003.17(4):438-42.
21. O'Donovan PJ and Livingston DM, BRCA1 and BRCA2:breast/ovarian cancer susceptibility gene products and participants in DNA double-strand break repair [J]. Carcinogenesis,2010.31(6):961-7.
22. Mucci LA, Wedren S, Tamimi RM, et al., The role of gene-environment interaction in the aetiology of human cancer:examples from cancers of the large bowel, lung and breast[J]. J Intern Med,2001.249(6):477-93.
23. Tempfer CB, Hefler LA, Schneeberger C, et al., How valid is single nucleotide polymorphism (SNP) diagnosis for the individual risk assessment of breast cancer?[J]. Gynecol Endocrinol,2006.22(3):155-9.
24. Ross JR, Saunders Y, Edmonds PM, et al.,Systematic review of role of bisphosphonates on skeletal morbidity in metastatic cancer[J]. BMJ,2003. 327(7413):469.
25. Eidtmann H, de Boer R, Bundred N, et al., Efficacy of zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole: 36-month results of the ZO-FAST Study [J]. Annals of Oncology,2010:
26. Gnant M, Mlineritsch B, Luschin-Ebengreuth G, et al., Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer:5-year follow-up of the ABCSG-12 bone-mineral density substudy[J]. Lancet Oncol,2008.9(9):840-9.
27. Tamoxifen for early breast cancer:an overview of the randomised trials. Early Breast Cancer Trialists' Collaborative Group[J]. Lancet,1998.351(9114): 1451-67.
28. Ring A and Dowsett M, Mechanisms of tamoxifen resistance [J]. Endocr Relat Cancer,2004.11(4):643-58.
29. Ali S and Coombes RC, Endocrine-responsive breast cancer and strategies for combating resistance [J]. Nat Rev Cancer,2002.2(2):101-12.
30. Osborne CK and Schiff R, Estrogen-receptor biology:continuing progress and therapeutic implications[J]. J Clin Oncol,2005.23(8):1616-22.
31. Veeman MT, Axelrod JD, and Moon RT, A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling[J]. Dev Cell,2003. 5(3):367-77.
32. Kikuchi A, Kishida S, and Yamamoto H, Regulation of Wnt signaling by protein-protein interaction and post-translational modifications [J]. Exp Mol Med,2006.38(1):1-10.
33. Katoh M, WNT/PCP signaling pathway and human cancer (review)[J]. Oncol Rep,2005.14(6):1583-8.
34. Brennan KR and Brown AM, Wnt proteins in mammary development and cancer[J]. J Mammary Gland Biol Neoplasia,2004.9(2):119-31.
35. Neville-Webbe HL, Evans CA, Coleman RE, et al.,Mechanisms of the synergistic interaction between the bisphosphonate zoledronic acid and the chemotherapy agent paclitaxel in breast cancer cells in vitro[J]. Tumour Biol, 2006.27(2):92-103.
36. Yan T, Yin W, Zhou Q, et al., The efficacy of zoledronic acid in breast cancer adjuvant therapy:a meta-analysis of randomised controlled trials[J]. Eur J Cancer,2012.48(2):187-95.
37. Ying J, Li H, Yu J, et al., WNT5A exhibits tumor-suppressive activity through antagonizing the Wnt/beta-catenin signaling, and is frequently methylated in colorectal cancer[J]. Clin Cancer Res,2008.14(1):55-61.
38. Kremenevskaja N, von Wasielewski R, Rao AS, et al., Wnt-5a has tumor suppressor activity in thyroid carcinoma[J]. Oncogene,2005.24(13):2144-54.
39. Topol L, Jiang X, Choi H, et al., Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation[J]. J Cell Biol, 2003.162(5):899-908.
40. Olson DJ and Gibo DM, Antisense wnt-5a mimics wnt-1-mediated C57MG mammary epithelial cell transformation[J]. Exp Cell Res,1998.241(1): 134-41.
41. Croce CM, Causes and consequences of microRNA dysregulation in cancer [J]. Nat Rev Genet,2009.10(10):704-14.
42. O'Day E and Lal A, MicroRNAs and their target gene networks in breast cancer[J]. Breast Cancer Res,2010.12(2):201.
43. Adams BD, Furneaux H, and White BA, The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-alpha (ERalpha) and represses ERalpha messenger RNA and protein expression in breast cancer cell lines[J]. Mol Endocrinol,2007.21(5):1132-47.
44. Li X and Carthew RW, A microRNA mediates EGF receptor signaling and promotes photoreceptor differentiation in the Drosophila eye[J]. Cell,2005. 123(7):1267-77.
45. Fazi F, Rosa A, Fatica A, et al., A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis[J]. Cell,2005.123(5):819-31.
46. Bock O and Felsenberg D, Bisphosphonates in the management of postmenopausal osteoporosis--optimizing efficacy in clinical practice [J]. Clin Interv Aging,2008.3(2):279-97.
47. Hillner BE, Ingle JN, Berenson JR,et al., American Society of Clinical Oncology guideline on the role of bisphosphonates in breast cancer. American Society of Clinical Oncology Bisphosphonates Expert Panel[J]. J Clin Oncol, 2000.18(6):1378-91.
48. Holen I and Coleman RE, Bisphosphonates as treatment of bone metastases[J]. Curr Pharm Des,2010.16(11):1262-71.
49. Gnant M, Mlineritsch B, Stoeger H, et al., Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer: 62-month follow-up from the ABCSG-12 randomised trial[J]. Lancet Oncol, 2011.12(7):631-41.
50. Eidtmann H, de Boer R, Bundred N, et al., Efficacy of zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole: 36-month results of the ZO-FAST Study [J]. Ann Oncol,2010.21(11): 2188-94.
51. Neville-Webbe HL, Rostami-Hodjegan A, Evans CA, et al., Sequence- and schedule-dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells[J]. Int J Cancer,2005.113(3): 364-71.
52. Kabeya Y, Mizushima N, Ueno T, et al., LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing [J]. EMBO J,2000.19(21):5720-8.
53. Holt SV, Wyspianska B, Randall KJ, et al., The development of an immunohistochemical method to detect the autophagy-associated protein LC3-Ⅱ in human tumor xenografts[J]. Toxicol Pathol,2011.39(3):516-23.
54. Wolff TA and Wilson JE, Genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility [J]. Am Fam Physician,2006. 74(10):1759-60.
55. Nelson HD, Huffman LH, Fu R, et al., Genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility:systematic evidence review for the U.S. Preventive Services Task Force [J]. Ann Intern Med,2005.143(5):362-79.
56. Li WF, Hu Z, Rao NY, et al., The prevalence of BRCA1 and BRCA2 germline mutations in high-risk breast cancer patients of Chinese Han nationality:two recurrent mutations were identified [J]. Breast Cancer Res Treat,2008.110(1): 99-109.
57. Roeder K and Luca D, Searching for disease susceptibility variants in structured populations [J]. Genomics,2009.93(1):1-4.
58. Pattaro C, Ruczinski I, Fallin DM, et al., Haplotype block partitioning as a tool for dimensionality reduction in SNP association studies[J]. BMC Genomics, 2008.9:405.
59. Feuk L, Marshall CR, Wintle RF, et al., Structural variants:changing the landscape of chromosomes and design of disease studies[J]. Hum Mol Genet, 2006.15 Spec No 1:R57-66.
60. Giacomini KM, Brett CM, Altman RB, et al., The pharmacogenetics research network:from SNP discovery to clinical drug response[J]. Clin Pharmacol Ther,2007.81(3):328-45.
61. Deloukas P and Bentley D, The HapMap project and its application to genetic studies of drug response[J]. Pharmacogenomics J,2004.4(2):88-90.
62. Giancola S, McKhann HI, Berard A, et al., Utilization of the three high-throughput SNP genotyping methods, the GOOD assay, Amplifluor and TaqMan, in diploid and polyploid plants[J]. Theor Appl Genet,2006.112(6): 1115-24.
63. Hopper JL, Genetic epidemiology of female breast cancer[J]. Semin Cancer Biol,2001.11(5):367-74.
64. Ma CX, Adjei AA, Salavaggione OE, et al., Human aromatase:gene resequencing and functional genomics[J]. Cancer Res,2005.65(23): 11071-82.
65. Barroso E, Milne RL, Fernandez LP, et al., FANCD2 associated with sporadic breast cancer risk[J]. Carcinogenesis,2006.27(9):1930-7.
66. Lejeune S, Huguet EL, Hamby A, et al., Wnt5a cloning, expression, and up-regulation in human primary breast cancers[J]. Clin Cancer Res,1995.1(2): 215-22.
67. Olson DJ, Gibo DM, Saggers G, et al., Reversion of uroepithelial cell tumorigenesis by the ectopic expression of human wnt-5a[J]. Cell Growth Differ,1997.8(4):417-23.
68. Mayo O, A century of Hardy-Weinberg equilibrium[J]. Twin Res Hum Genet, 2008.11(3):249-56.
69. Cui X, Schiff R, Arpino G, et al., Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy [J]. J Clin Oncol,2005. 23(30):7721-35.
70. Allred DC,Brown P, and Medina D, The origins of estrogen receptor alpha-positive and estrogen receptor alpha-negative human breast cancer[J]. Breast Cancer Res,2004.6(6):240-5.
71. Dowsett M, Cuzick J,Wale C, et al., Retrospective analysis of time to recurrence in the ATAC trial according to hormone receptor status:an hypothesis-generating study[J]. J Clin Oncol,2005.23(30):7512-7.
72. Bardou VJ, Arpino G, Elledge RM, et al., Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases [J]. J Clin Oncol,2003.21(10):1973-9.
73. Elledge RM, Green S, Pugh R, et al., Estrogen receptor (ER) and progesterone receptor (PgR), by ligand-binding assay compared with ER, PgR and pS2, by immuno-histochemistry in predicting response to tamoxifen in metastatic breast cancer:a Southwest Oncology Group Study[J]. Int J Cancer,2000. 89(2):111-7.
74. Arpino G, Weiss H, Lee AV, et al., Estrogen receptor-positive, progesterone receptor-negative breast cancer:association with growth factor receptor expression and tamoxifen resistance[J]. J Natl Cancer Inst,2005.97(17): 1254-61.
75. Creighton CJ, Kent Osborne C, van de Vijver MJ, et al., Molecular profiles of progesterone receptor loss in human breast tumors[J]. Breast Cancer Res Treat, 2009.114(2):287-99.
76. Oishi I, Suzuki H, Onishi N, et al., The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway [J]. Genes Cells, 2003.8(7):645-54.
77. Reguart N, He B, Taron M, et al., The role of Wnt signaling in cancer and stem cells[J]. Future Oncol,2005.1(6):787-97.
78. McDonald SL and Silver A, The opposing roles of Wnt-5a in cancer[J]. Br J Cancer,2009.101(2):209-14.
79. Da Forno PD, Pringle JH, Hutchinson P, et al., WNT5A expression increases during melanoma progression and correlates with outcome[J]. Clin Cancer Res, 2008.14(18):5825-32.
80. Wang Q, Williamson M, Bott S, et al., Hypomethylation of WNT5A, CRIP1 and S100P in prostate cancer[J]. Oncogene,2007.26(45):6560-5.
81. Roman-Gomez J, Jimenez-Velasco A, Cordeu L, et al., WNT5A, a putative tumour suppressor of lymphoid malignancies, is inactivated by aberrant methylation in acute lymphoblastic leukaemia[J]. Eur J Cancer,2007.43(18): 2736-46.
82. Fernandez-Cobo M, Zammarchi F, Mandeli J, et al., Expression of Wnt5A and Wnt10B in non-immortalized breast cancer cells[J]. Oncol Rep.2007.17(4): 903-7.
83. Zhang S, Chen L, Cui B, et al.,ROR1 Is Expressed in Human Breast Cancer and Associated with Enhanced Tumor-Cell Growth[J]. PLoS One,2012.7(3): e31127.
84. Yang X, Phillips DL, Ferguson AT, et al., Synergistic activation of functional estrogen receptor (ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells [J]. Cancer Res,2001.61(19):7025-9.
85. Keen JC, Yan L, Mack KM, et al., A novel histone deacetylase inhibitor, scriptaid, enhances expression of functional estrogen receptor alpha (ER) in ER negative human breast cancer cells in combination with 5-aza 2'-deoxycytidine[J]. Breast Cancer Res Treat,2003.81(3):177-86.
86. Yang X, Ferguson AT, Nass SJ, et al., Transcriptional activation of estrogen receptor alpha in human breast cancer cells by histone deacetylase inhibition[J]. Cancer Res,2000.60(24):6890-4.
87. Yoshimoto N, Toyama T, Takahashi S, et al., Distinct expressions of microRNAs that directly target estrogen receptor alpha in human breast cancer[J]. Breast Cancer Res Treat,2011.130(1):331-9.
88. Pandey DP and Picard D, miR-22 inhibits estrogen signaling by directly targeting the estrogen receptor alpha mRNA[J]. Mol Cell Biol,2009.29(13): 3783-90.
89. Zhao JJ, Lin J, Yang H, et al., MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer[J]. J Biol Chem,2008.283(45):31079-86.
90. Hossain A, Kuo MT,and Saunders GF, Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA[J]. Mol Cell Biol,2006. 26(21):8191-201.
91. Foley NH, Bray I, Watters KM, et al., MicroRNAs 10a and 10b are potent inducers of neuroblastoma cell differentiation through targeting of nuclear receptor corepressor 2[J]. Cell Death Differ,2011.18(7):1089-98.
92. Leris AC, Roberts TR, Jiang WG, et al., WNT5A expression in human breast cancer[J]. Anticancer Res,2005.25(2A):731-4.
93. Roarty K, Baxley SE, Crowley MR, et al, Loss of TGF-beta or Wnt5a results in an increase in Wnt/beta-catenin activity and redirects mammary tumour phenotype[J]. Breast Cancer Res,2009.11(2):R19.
94. Dejmek J, Leandersson K, Manjer J, et al., Expression and signaling activity of Wnt-5a/discoidin domain receptor-1 and Syk plays distinct but decisive roles in breast cancer patient survival [J]. Clin Cancer Res,2005.11(2 Pt 1): 520-8.
95. Yin W, Di G, Zhou L, et al., Time-varying pattern of recurrence risk for Chinese breast cancer patients[J]. Breast Cancer Res Treat,2009.114(3): 527-35.
96. Normanno N, Di Maio M, De Maio E, et al., Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer [J]. Endocr Relat Cancer,2005.12(4):721-47.
97. Musgrove EA and Sutherland RL, Biological determinants of endocrine resistance in breast cancer[J]. Nat Rev Cancer,2009.9(9):631-43.
98. Clarke R, Leonessa F, Welch JN, et al., Cellular and molecular pharmacology of antiestrogen action and resistance[J]. Pharmacol Rev,2001.53(1):25-71.
99. Zhou L, Yan T, Jiang Y, et al., Prognostic and predictive value of TFF1 for adjuvant endocrine therapy in Chinese women with early ER positive breast cancer:comparing aromatase inhibitors with tamoxifen[J]. Breast,2011.20(1): 15-20.
1. Roodman GD. Mechanisms of disease:Mechanisms of bone metastasis[J]. N Engl J Med,2004,350(16):1655-1664.
2. Coleman RE. Metastatic bone disease:clinical features, pathophysiology and treatment strategies [J]. Cancer Treat Rev,2001,27(3):165-176.
3. Saad F, Lipton A, Cook R, et al. Pathologic fractures correlate with reduced survival in patients with malignant bone disease[J]. Cancer,2007,110: 1860-1867.
4. Ross JR, Saunders Y, Edmonds PM, et al. Systematic review of role of bisphosphonates on skeletal morbidity in metastatic cancer[J]. BMJ, 2003,327(7413):469-472.
5. Green JR. Bisphosphonates:Preclinical review[J]. Oncologist,2004,9:3-13.
6. Coleman RE, Bisphosphonates:Clinical experience[J]. Oncologist,2004,9: 14-27.
7. Wood J, Bonjean K, Ruetz S, et al. Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid[J]. J Pharmacol Exp Ther,2002, 302(3):1055-1061.
8. Nicosia RE What is the role of vascular endothelial growth factor-related molecules in tumor angiogenesis?[J]. Am J Pathol,1998,153(1):11-16.
9. Santini D, Vincenzi B, Galluzzo S, et al. Repeated intermittent low-dose therapy with zoledronic acid induces an early, sustained, and long-lasting decrease of peripheral vascular endothelial growth factor levels in cancer patients[J]. Clin Cancer Res,2007,13(15):4482-4486.
10. Vincenzi B, Santini D, DicuonzoI G, et al. Zoledronic acid-related angiogenesis modifications and survival in advanced breast cancer patients[J]. J Interferon Cytokine Res,2005,25(3):144-151.
11. Verdijk R, Franke HR, Wolbers F, et al. Differential effects of bisphosphonates on breast cancer cell lines[J]. Cancer Lett,2007,246(1-2):308-312.
12. Neville-webbe HL, Evans CA, Coleman RE, et al. Mechanisms of the synergistic interaction between the bisphosphonate zoledronic acid and the chemotherapy agent paclitaxel in breast cancer cells in vitro[J]. Tumor Biol, 2006,27(2):92-103.
13. Ottewell PD, Monkkonen H, Jones M, et al. Antitumor effects of doxorubicin followed by zoledronic acid in a mouse model of breast cancer[J]. J Natl Cancer Inst,2008,100(16):1167-1178.
14. Neville-webbe HL, Rostami-hojegan A, Evans CA, et al. Sequence-and schedule-dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells[J]. Int J Cancer,2005,113(3): 364-371.
15. Ottewell PD, Deux B, Monkkonen H, et al. Differential effect of doxorubicin and zoledronic acid on intraosseous versus extraosseous breast tumor growth in vivo[J]. Clin Cancer Res,2008,14(14):4658-4666.
16. Winter MC, Thorpe HC, Burkinshaw R, et al. Presentation title:The addition of zoledronic acid to neoadjuvant chemotherapy may influence pathological response exploratory evidence for direct anti-tumor activity in breast cancer. In:31th annual San Antonio breast cancer symposium,2008, abstract 5101.
17. Powles T, Paterson A, Mccloskey E, et al. Reduction in bone relapse and improved survival with oral clodronate for adjuvant treatment of operable breast cancer [ISRCTN83688026][J]. Breast Cancer Res,2006,8(2):R 13
18. Saarto T, Blpmqvist C, Virkkunen P, et al. Adjuvant clodronate treatment does not reduce the frequency of skeletal metastases in node-positive breast cancer patients:5-year results of a randomized controlled trial [J]. J Clin Oncol,2001, 19(1):10-17.
19. Saarto T, Vehmanen L, Blomqvist C, et al. Ten-year follow-up of a randomized controlled trial of adjuvant clodronate treatment in node-positive breast cancer patients[J]. J Clin Oncol.2004.22(14):9S-9S.
20. Crivellari D, Sun Z. Coates AS, et al. Letrozole compared with tamoxifen for elderly patients with endocrine-responsive early breast cancer:The BIG 1-98 trial[J]. J Clin Oncol,2008.26(12):1972-1979.
21. Coleman RE, Body JJ, Gralow JR, et al. Bone loss in patients with breast cancer receiving aromatase inhibitors and associated treatment strategies [J]. Cancer Treat Rev,2008,34:S31-S42.
22. Bundred NJ, Campbell ID, Davidson N, et al. Effective inhibition of aromatase inhibitor-associated bone loss by zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole[J]. Cancer,2008, 112(5):1001-1010.
23. Eidtmann H, Bundered NJ, Deboer R, et al. Presentation title:The Effect of Zoledronic Acid on Aromatase Inhibitor-Associated Bone Loss in Postmenopausal Women With Early Breast Cancer Receiving Adjuvant Letrozole:36 Months Follow-Up of ZO-FAST. SAN ANTONIO,2008: abstract 44.
24. Gnant M, Mlineritsch B, Luschin-Ebengreuth G, et al. Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer:5-year follow-up of the ABCSG-12 bone-mineral density substudy[J]. Lancet Oncol,2008,9(9):840-849.
25. Gnant M, Mlineritsch B, Schippinger W, et al. Adjuvant ovarian suppression combined with tamoxifen or anastrozole, alone or in combination with zoledronic acid, in premenopausal women with hormone-responsive, stage I and II breast cancer:First effi cacy results from ABCSG-12[J]. J Clin Oncol, ASCO Annual Meeting Proceedings 2008.26:abstract LBA4.
26. Bilezikian JP. Osteonecrosis of the jaw-Do biphosphonates pose a risk?[J]. N Engl J Med,2006,355(22):2278-2281.
1. Hopper-Borge E, Xu X, Shen T, et al. Human multidrug resistance protein 7 (ABCC10) is a resistance factor for nucleoside analogues and epothilone B[J]. Cancer Res,2009,69(1):178-184.
2. Galletti E, Magnani M. Renzulli ML. et al. Paclitaxel and docetaxel resistance: molecular mechanisms and development of new generation taxanes[J]. ChemMedChem,2007,2(7):920-942.
3. Rottenberg S, Nygren AO, Pajic M, et al. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer[J]. Proc Natl Acad Sci USA,2007,104(29):12117-12122.
4. Maeno K, Ito K, Hama Y, et al. Mutation of the class Ⅰ beta-tubulin gene does not predict response to paclitaxel for breast cancer[J]. Cancer Lett,2003, 198(1):89-97.
5. Kamath K, Wilson L, Cabral F, et al. BetaⅢ-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability [J]. J Biol Chem,2005,280(13):12902-12907.
6. Tommasi S, Mangia A, Lacalamita R, et al. Cytoskeleton and paclitaxel sensitivity in breast cancer:the role of beta-tubulins[J]. Int J Cancer,2007, 120(10):2078-2085.
7. Rouzier R, Rajan R, Wagner P, et al. Microtubule-associated protein tau:a marker of paclitaxel sensitivity in breast cancer[J]. Proc Natl Acad Sci USA, 2005,102(23):8315-8320.
8. Andre F, Hatzis C, Anderson K, et al. Microtubule-associated protein-tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in estrogen receptor-positive breast cancer[J].Clin Cancer Res,2007,13(7): 2061-2067.
9. Gril B, Vidal M, Assayag F, et al. Grb2-SH3 ligand inhibits the growth of HER2+cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel[J]. Int J Cancer,2007,121(2): 407-415.
10. Konecny GE, Thomssen C, Luck HJ, et al. Her-2/neu gene amplification and response to paclitaxel in patients with metastatic breast cancer[J]. J Natl Cancer Inst,2004,96(15):1141-1151.
11. Chen SC, Chang HK, Lin YC, et al. High pathologic complete response in HER 2-positive locally advanced breast cancer after primary systemic chemotherapy with weekly docetaxel and epirubicin[J]. Jpn J Clin Oncol, 2008,38(2):99-105.
12. Andre F, Mazouni C, Liedtke C, et al. HER2 expression and efficacy of preoperative paclitaxel/FAC chemotherapy in breast cancer[J]. Breast Cancer Res Treat,2008,108(2):183-190.
13. Estevez LG, Cuevas JM, Anton A, et al. Weekly docetaxel as neoadjuvant chemotherapy for stage Ⅱ and Ⅲ breast cancer:efficacy and correlation with biological markers in a phase Ⅱ. multicenter study[J]. Clin Cancer Res,2003, 9(2):686-692.
14. Tham YL, Gomez LF, Mohsin S, et al. Clinical response to neoadjuvant docetaxel predicts improved outcome in patients with large locally advanced breast cancers[J]. Breast Cancer Res Treat,2005,94(3):279-284.
15. Kim SJ, Miyoshi Y, Taguchi T, et al. High thioredoxin expression is associated with resistance to docetaxel in primary breast cancer[J]. Clin Cancer Res, 2005,11(23):8425-8430.
16. Nakayama S, Torikoshi Y, Takahashi T, et al. Prediction of paclitaxel sensitivity by CDK1 and CDK2 activity in human breast cancer cells[J]. Breast Cancer Res,2009,11(1):R12.
17. Vredenburg MR, Ojima I, Veith J, et al. Effects of orally active taxanes on P-glycoprotein modulation and colon and breast carcinoma drug resistance[J]. J Natl Cancer Inst,2001,93(16):1234-1245.
18. Patil Y, Sadhukha T, Ma L, et al. Nanoparticle-mediated simultaneous and targeted delivery of paclitaxel and tariquidar overcomes tumor drug resistance[J]. J Control Release,2009,136(1):21-29.
19. Mi Y and Lou L ZD6474 reverses multidrug resistance by directly inhibiting the function of P-glycoprotein[J]. Br J Cancer,2007,97(7):934-940.
20. Ferlini C, Raspaglio G, Mozzetti S, et al. The seco-taxane IDN5390 is able to target class III beta-tubulin and to overcome paclitaxel resistance[J].Cancer Res,2005,65(6):2397-2405.
21. Kim SN, Kim NH, Park YS, et al.7-Diethylamino-3(2'-benzoxazolyl) coumarin is a novel microtubule inhibitor with antimitotic activity in multidrug resistant cancer cells[J]. Biochem Pharmacol,2009,77(12): 1773-1779.
22. Pegram MD, Konecny GE, O'Callaghan C, et al. Rational combinations of trastuzumab with chemotherapeutic drugs used in the treatment of breast cancer[J]. J Natl Cancer Inst,2004,96(10):739-749.
23. Crown J, O'Leary M, and Ooi WS Docetaxel and paclitaxel in the treatment of breast cancer:a review of clinical experience [J]. Oncologist,2004,9 (Suppl 2):24-32.
24. Ting HJ, Hsu J, Bao BY, et al. Docetaxel-induced growth inhibition and apoptosis in androgen independent prostate cancer cells are enhanced by 1alpha,25-dihydroxyvitamin D3[J]. Cancer Lett,2007,247(1):122-129.
25. Marshall J, Chen H, Yang D, et al. A phase Ⅰ trial of a Bcl-2 antisense (G3139) and weekly docetaxel in patients with advanced breast cancer and other solid tumors[J]. Ann Oncol,2004,15(8):1274-1283.
2. Anderson WF, Chatterjee N, Ershler WB, et al., Estrogen receptor breast cancer phenotypes in the Surveillance, Epidemiology, and End Results database[J]. Breast Cancer Res Treat,2002.76(1):27-36.
3. Murphy LC and Watson P, Steroid receptors in human breast tumorigenesis and breast cancer progression[J]. Biomed Pharmacother,2002.56(2):65-77.
4. Dunnwald LK, Rossing MA, and Li CI, Hormone receptor status, tumor characteristics, and prognosis:a prospective cohort of breast cancer patients[J]. Breast Cancer Res,2007.9(1):R6.
5. Johnston SR, Acquired tamoxifen resistance in human breast cancer--potential mechanisms and clinical implications[J]. Anticancer Drugs,1997.8(10): 911-30.
6. Logan CY and Nusse R, The Wnt signaling pathway in development and disease[J]. Annu Rev Cell Dev Biol,2004.20:781-810.
7. Peifer M and Polakis P, Wnt signaling in oncogenesis and embryogenesis--a look outside the nucleus[J]. Science,2000.287(5458):1606-9.
8. Kuhl M, Sheldahl LC, Park M, et al., The Wnt/Ca2+pathway:a new vertebrate Wnt signaling pathway takes shape [J]. Trends Genet,2000.16(7): 279-83.
9. Howng SL, Wu CH, Cheng TS, et al., Differential expression of Wnt genes, beta-catenin and E-cadherin in human brain tumors[J]. Cancer Lett,2002. 183(1):95-101.
10. Weeraratna AT, Jiang Y, Hostetter G, et al., Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma[J]. Cancer Cell,2002.1(3): 279-88.
11. Kurayoshi M, Oue N, Yamamoto H, et al., Expression of Wnt-5a is correlated with aggressiveness of gastric cancer by stimulating cell migration and invasion[J]. Cancer Res,2006.66(21):10439-48.
12. Ripka S, Konig A, Buchholz M, et al., WNT5A--target of CUTL1 and potent modulator of tumor cell migration and invasion in pancreatic cancer [J]. Carcinogenesis,2007.28(6):1178-87.
13. Liang H, Chen Q, Coles AH, et al., Wnt5a inhibits B cell proliferation and functions as a tumor suppressor in hematopoietic tissue[J]. Cancer Cell,2003. 4(5):349-60.
14. Dejmek J, Dejmek A, Safholm A, et al., Wnt-5a protein expression in primary dukes B colon cancers identifies a subgroup of patients with good prognosis [J]. Cancer Res,2005.65(20):9142-6.
15. Jonsson M, Dejmek J, Bendahl PO, et al., Loss of Wnt-5a protein is associated with early relapse in invasive ductal breast carcinomas[J]. Cancer Res,2002. 62(2):409-16.
16. Ford CE, Ekstrom EJ, and Andersson T, Wnt-5a signaling restores tamoxifen sensitivity in estrogen receptor-negative breast cancer cells[J]. Proc Natl Acad Sci USA,2009.106(10):3919-24.
17. Bartel DP, MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell,2004.116(2):281-97.
18. Lee RC, Feinbaum RL, and Ambros V, The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J]. Cell, 1993.75(5):843-54.
19. Wightman B, Ha I, and Ruvkun G, Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans[J]. Cell,1993.75(5):855-62.
20. Doench JG, Petersen CP, and Sharp PA, siRNAs can function as miRNAs[J]. Genes Dev,2003.17(4):438-42.
21. O'Donovan PJ and Livingston DM, BRCA1 and BRCA2:breast/ovarian cancer susceptibility gene products and participants in DNA double-strand break repair [J]. Carcinogenesis,2010.31(6):961-7.
22. Mucci LA, Wedren S, Tamimi RM, et al., The role of gene-environment interaction in the aetiology of human cancer:examples from cancers of the large bowel, lung and breast[J]. J Intern Med,2001.249(6):477-93.
23. Tempfer CB, Hefler LA, Schneeberger C, et al., How valid is single nucleotide polymorphism (SNP) diagnosis for the individual risk assessment of breast cancer?[J]. Gynecol Endocrinol,2006.22(3):155-9.
24. Ross JR, Saunders Y, Edmonds PM, et al.,Systematic review of role of bisphosphonates on skeletal morbidity in metastatic cancer[J]. BMJ,2003. 327(7413):469.
25. Eidtmann H, de Boer R, Bundred N, et al., Efficacy of zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole: 36-month results of the ZO-FAST Study [J]. Annals of Oncology,2010:
26. Gnant M, Mlineritsch B, Luschin-Ebengreuth G, et al., Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer:5-year follow-up of the ABCSG-12 bone-mineral density substudy[J]. Lancet Oncol,2008.9(9):840-9.
27. Tamoxifen for early breast cancer:an overview of the randomised trials. Early Breast Cancer Trialists' Collaborative Group[J]. Lancet,1998.351(9114): 1451-67.
28. Ring A and Dowsett M, Mechanisms of tamoxifen resistance [J]. Endocr Relat Cancer,2004.11(4):643-58.
29. Ali S and Coombes RC, Endocrine-responsive breast cancer and strategies for combating resistance [J]. Nat Rev Cancer,2002.2(2):101-12.
30. Osborne CK and Schiff R, Estrogen-receptor biology:continuing progress and therapeutic implications[J]. J Clin Oncol,2005.23(8):1616-22.
31. Veeman MT, Axelrod JD, and Moon RT, A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling[J]. Dev Cell,2003. 5(3):367-77.
32. Kikuchi A, Kishida S, and Yamamoto H, Regulation of Wnt signaling by protein-protein interaction and post-translational modifications [J]. Exp Mol Med,2006.38(1):1-10.
33. Katoh M, WNT/PCP signaling pathway and human cancer (review)[J]. Oncol Rep,2005.14(6):1583-8.
34. Brennan KR and Brown AM, Wnt proteins in mammary development and cancer[J]. J Mammary Gland Biol Neoplasia,2004.9(2):119-31.
35. Neville-Webbe HL, Evans CA, Coleman RE, et al.,Mechanisms of the synergistic interaction between the bisphosphonate zoledronic acid and the chemotherapy agent paclitaxel in breast cancer cells in vitro[J]. Tumour Biol, 2006.27(2):92-103.
36. Yan T, Yin W, Zhou Q, et al., The efficacy of zoledronic acid in breast cancer adjuvant therapy:a meta-analysis of randomised controlled trials[J]. Eur J Cancer,2012.48(2):187-95.
37. Ying J, Li H, Yu J, et al., WNT5A exhibits tumor-suppressive activity through antagonizing the Wnt/beta-catenin signaling, and is frequently methylated in colorectal cancer[J]. Clin Cancer Res,2008.14(1):55-61.
38. Kremenevskaja N, von Wasielewski R, Rao AS, et al., Wnt-5a has tumor suppressor activity in thyroid carcinoma[J]. Oncogene,2005.24(13):2144-54.
39. Topol L, Jiang X, Choi H, et al., Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation[J]. J Cell Biol, 2003.162(5):899-908.
40. Olson DJ and Gibo DM, Antisense wnt-5a mimics wnt-1-mediated C57MG mammary epithelial cell transformation[J]. Exp Cell Res,1998.241(1): 134-41.
41. Croce CM, Causes and consequences of microRNA dysregulation in cancer [J]. Nat Rev Genet,2009.10(10):704-14.
42. O'Day E and Lal A, MicroRNAs and their target gene networks in breast cancer[J]. Breast Cancer Res,2010.12(2):201.
43. Adams BD, Furneaux H, and White BA, The micro-ribonucleic acid (miRNA) miR-206 targets the human estrogen receptor-alpha (ERalpha) and represses ERalpha messenger RNA and protein expression in breast cancer cell lines[J]. Mol Endocrinol,2007.21(5):1132-47.
44. Li X and Carthew RW, A microRNA mediates EGF receptor signaling and promotes photoreceptor differentiation in the Drosophila eye[J]. Cell,2005. 123(7):1267-77.
45. Fazi F, Rosa A, Fatica A, et al., A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis[J]. Cell,2005.123(5):819-31.
46. Bock O and Felsenberg D, Bisphosphonates in the management of postmenopausal osteoporosis--optimizing efficacy in clinical practice [J]. Clin Interv Aging,2008.3(2):279-97.
47. Hillner BE, Ingle JN, Berenson JR,et al., American Society of Clinical Oncology guideline on the role of bisphosphonates in breast cancer. American Society of Clinical Oncology Bisphosphonates Expert Panel[J]. J Clin Oncol, 2000.18(6):1378-91.
48. Holen I and Coleman RE, Bisphosphonates as treatment of bone metastases[J]. Curr Pharm Des,2010.16(11):1262-71.
49. Gnant M, Mlineritsch B, Stoeger H, et al., Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer: 62-month follow-up from the ABCSG-12 randomised trial[J]. Lancet Oncol, 2011.12(7):631-41.
50. Eidtmann H, de Boer R, Bundred N, et al., Efficacy of zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole: 36-month results of the ZO-FAST Study [J]. Ann Oncol,2010.21(11): 2188-94.
51. Neville-Webbe HL, Rostami-Hodjegan A, Evans CA, et al., Sequence- and schedule-dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells[J]. Int J Cancer,2005.113(3): 364-71.
52. Kabeya Y, Mizushima N, Ueno T, et al., LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing [J]. EMBO J,2000.19(21):5720-8.
53. Holt SV, Wyspianska B, Randall KJ, et al., The development of an immunohistochemical method to detect the autophagy-associated protein LC3-Ⅱ in human tumor xenografts[J]. Toxicol Pathol,2011.39(3):516-23.
54. Wolff TA and Wilson JE, Genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility [J]. Am Fam Physician,2006. 74(10):1759-60.
55. Nelson HD, Huffman LH, Fu R, et al., Genetic risk assessment and BRCA mutation testing for breast and ovarian cancer susceptibility:systematic evidence review for the U.S. Preventive Services Task Force [J]. Ann Intern Med,2005.143(5):362-79.
56. Li WF, Hu Z, Rao NY, et al., The prevalence of BRCA1 and BRCA2 germline mutations in high-risk breast cancer patients of Chinese Han nationality:two recurrent mutations were identified [J]. Breast Cancer Res Treat,2008.110(1): 99-109.
57. Roeder K and Luca D, Searching for disease susceptibility variants in structured populations [J]. Genomics,2009.93(1):1-4.
58. Pattaro C, Ruczinski I, Fallin DM, et al., Haplotype block partitioning as a tool for dimensionality reduction in SNP association studies[J]. BMC Genomics, 2008.9:405.
59. Feuk L, Marshall CR, Wintle RF, et al., Structural variants:changing the landscape of chromosomes and design of disease studies[J]. Hum Mol Genet, 2006.15 Spec No 1:R57-66.
60. Giacomini KM, Brett CM, Altman RB, et al., The pharmacogenetics research network:from SNP discovery to clinical drug response[J]. Clin Pharmacol Ther,2007.81(3):328-45.
61. Deloukas P and Bentley D, The HapMap project and its application to genetic studies of drug response[J]. Pharmacogenomics J,2004.4(2):88-90.
62. Giancola S, McKhann HI, Berard A, et al., Utilization of the three high-throughput SNP genotyping methods, the GOOD assay, Amplifluor and TaqMan, in diploid and polyploid plants[J]. Theor Appl Genet,2006.112(6): 1115-24.
63. Hopper JL, Genetic epidemiology of female breast cancer[J]. Semin Cancer Biol,2001.11(5):367-74.
64. Ma CX, Adjei AA, Salavaggione OE, et al., Human aromatase:gene resequencing and functional genomics[J]. Cancer Res,2005.65(23): 11071-82.
65. Barroso E, Milne RL, Fernandez LP, et al., FANCD2 associated with sporadic breast cancer risk[J]. Carcinogenesis,2006.27(9):1930-7.
66. Lejeune S, Huguet EL, Hamby A, et al., Wnt5a cloning, expression, and up-regulation in human primary breast cancers[J]. Clin Cancer Res,1995.1(2): 215-22.
67. Olson DJ, Gibo DM, Saggers G, et al., Reversion of uroepithelial cell tumorigenesis by the ectopic expression of human wnt-5a[J]. Cell Growth Differ,1997.8(4):417-23.
68. Mayo O, A century of Hardy-Weinberg equilibrium[J]. Twin Res Hum Genet, 2008.11(3):249-56.
69. Cui X, Schiff R, Arpino G, et al., Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy [J]. J Clin Oncol,2005. 23(30):7721-35.
70. Allred DC,Brown P, and Medina D, The origins of estrogen receptor alpha-positive and estrogen receptor alpha-negative human breast cancer[J]. Breast Cancer Res,2004.6(6):240-5.
71. Dowsett M, Cuzick J,Wale C, et al., Retrospective analysis of time to recurrence in the ATAC trial according to hormone receptor status:an hypothesis-generating study[J]. J Clin Oncol,2005.23(30):7512-7.
72. Bardou VJ, Arpino G, Elledge RM, et al., Progesterone receptor status significantly improves outcome prediction over estrogen receptor status alone for adjuvant endocrine therapy in two large breast cancer databases [J]. J Clin Oncol,2003.21(10):1973-9.
73. Elledge RM, Green S, Pugh R, et al., Estrogen receptor (ER) and progesterone receptor (PgR), by ligand-binding assay compared with ER, PgR and pS2, by immuno-histochemistry in predicting response to tamoxifen in metastatic breast cancer:a Southwest Oncology Group Study[J]. Int J Cancer,2000. 89(2):111-7.
74. Arpino G, Weiss H, Lee AV, et al., Estrogen receptor-positive, progesterone receptor-negative breast cancer:association with growth factor receptor expression and tamoxifen resistance[J]. J Natl Cancer Inst,2005.97(17): 1254-61.
75. Creighton CJ, Kent Osborne C, van de Vijver MJ, et al., Molecular profiles of progesterone receptor loss in human breast tumors[J]. Breast Cancer Res Treat, 2009.114(2):287-99.
76. Oishi I, Suzuki H, Onishi N, et al., The receptor tyrosine kinase Ror2 is involved in non-canonical Wnt5a/JNK signalling pathway [J]. Genes Cells, 2003.8(7):645-54.
77. Reguart N, He B, Taron M, et al., The role of Wnt signaling in cancer and stem cells[J]. Future Oncol,2005.1(6):787-97.
78. McDonald SL and Silver A, The opposing roles of Wnt-5a in cancer[J]. Br J Cancer,2009.101(2):209-14.
79. Da Forno PD, Pringle JH, Hutchinson P, et al., WNT5A expression increases during melanoma progression and correlates with outcome[J]. Clin Cancer Res, 2008.14(18):5825-32.
80. Wang Q, Williamson M, Bott S, et al., Hypomethylation of WNT5A, CRIP1 and S100P in prostate cancer[J]. Oncogene,2007.26(45):6560-5.
81. Roman-Gomez J, Jimenez-Velasco A, Cordeu L, et al., WNT5A, a putative tumour suppressor of lymphoid malignancies, is inactivated by aberrant methylation in acute lymphoblastic leukaemia[J]. Eur J Cancer,2007.43(18): 2736-46.
82. Fernandez-Cobo M, Zammarchi F, Mandeli J, et al., Expression of Wnt5A and Wnt10B in non-immortalized breast cancer cells[J]. Oncol Rep.2007.17(4): 903-7.
83. Zhang S, Chen L, Cui B, et al.,ROR1 Is Expressed in Human Breast Cancer and Associated with Enhanced Tumor-Cell Growth[J]. PLoS One,2012.7(3): e31127.
84. Yang X, Phillips DL, Ferguson AT, et al., Synergistic activation of functional estrogen receptor (ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells [J]. Cancer Res,2001.61(19):7025-9.
85. Keen JC, Yan L, Mack KM, et al., A novel histone deacetylase inhibitor, scriptaid, enhances expression of functional estrogen receptor alpha (ER) in ER negative human breast cancer cells in combination with 5-aza 2'-deoxycytidine[J]. Breast Cancer Res Treat,2003.81(3):177-86.
86. Yang X, Ferguson AT, Nass SJ, et al., Transcriptional activation of estrogen receptor alpha in human breast cancer cells by histone deacetylase inhibition[J]. Cancer Res,2000.60(24):6890-4.
87. Yoshimoto N, Toyama T, Takahashi S, et al., Distinct expressions of microRNAs that directly target estrogen receptor alpha in human breast cancer[J]. Breast Cancer Res Treat,2011.130(1):331-9.
88. Pandey DP and Picard D, miR-22 inhibits estrogen signaling by directly targeting the estrogen receptor alpha mRNA[J]. Mol Cell Biol,2009.29(13): 3783-90.
89. Zhao JJ, Lin J, Yang H, et al., MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer[J]. J Biol Chem,2008.283(45):31079-86.
90. Hossain A, Kuo MT,and Saunders GF, Mir-17-5p regulates breast cancer cell proliferation by inhibiting translation of AIB1 mRNA[J]. Mol Cell Biol,2006. 26(21):8191-201.
91. Foley NH, Bray I, Watters KM, et al., MicroRNAs 10a and 10b are potent inducers of neuroblastoma cell differentiation through targeting of nuclear receptor corepressor 2[J]. Cell Death Differ,2011.18(7):1089-98.
92. Leris AC, Roberts TR, Jiang WG, et al., WNT5A expression in human breast cancer[J]. Anticancer Res,2005.25(2A):731-4.
93. Roarty K, Baxley SE, Crowley MR, et al, Loss of TGF-beta or Wnt5a results in an increase in Wnt/beta-catenin activity and redirects mammary tumour phenotype[J]. Breast Cancer Res,2009.11(2):R19.
94. Dejmek J, Leandersson K, Manjer J, et al., Expression and signaling activity of Wnt-5a/discoidin domain receptor-1 and Syk plays distinct but decisive roles in breast cancer patient survival [J]. Clin Cancer Res,2005.11(2 Pt 1): 520-8.
95. Yin W, Di G, Zhou L, et al., Time-varying pattern of recurrence risk for Chinese breast cancer patients[J]. Breast Cancer Res Treat,2009.114(3): 527-35.
96. Normanno N, Di Maio M, De Maio E, et al., Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer [J]. Endocr Relat Cancer,2005.12(4):721-47.
97. Musgrove EA and Sutherland RL, Biological determinants of endocrine resistance in breast cancer[J]. Nat Rev Cancer,2009.9(9):631-43.
98. Clarke R, Leonessa F, Welch JN, et al., Cellular and molecular pharmacology of antiestrogen action and resistance[J]. Pharmacol Rev,2001.53(1):25-71.
99. Zhou L, Yan T, Jiang Y, et al., Prognostic and predictive value of TFF1 for adjuvant endocrine therapy in Chinese women with early ER positive breast cancer:comparing aromatase inhibitors with tamoxifen[J]. Breast,2011.20(1): 15-20.
1. Roodman GD. Mechanisms of disease:Mechanisms of bone metastasis[J]. N Engl J Med,2004,350(16):1655-1664.
2. Coleman RE. Metastatic bone disease:clinical features, pathophysiology and treatment strategies [J]. Cancer Treat Rev,2001,27(3):165-176.
3. Saad F, Lipton A, Cook R, et al. Pathologic fractures correlate with reduced survival in patients with malignant bone disease[J]. Cancer,2007,110: 1860-1867.
4. Ross JR, Saunders Y, Edmonds PM, et al. Systematic review of role of bisphosphonates on skeletal morbidity in metastatic cancer[J]. BMJ, 2003,327(7413):469-472.
5. Green JR. Bisphosphonates:Preclinical review[J]. Oncologist,2004,9:3-13.
6. Coleman RE, Bisphosphonates:Clinical experience[J]. Oncologist,2004,9: 14-27.
7. Wood J, Bonjean K, Ruetz S, et al. Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid[J]. J Pharmacol Exp Ther,2002, 302(3):1055-1061.
8. Nicosia RE What is the role of vascular endothelial growth factor-related molecules in tumor angiogenesis?[J]. Am J Pathol,1998,153(1):11-16.
9. Santini D, Vincenzi B, Galluzzo S, et al. Repeated intermittent low-dose therapy with zoledronic acid induces an early, sustained, and long-lasting decrease of peripheral vascular endothelial growth factor levels in cancer patients[J]. Clin Cancer Res,2007,13(15):4482-4486.
10. Vincenzi B, Santini D, DicuonzoI G, et al. Zoledronic acid-related angiogenesis modifications and survival in advanced breast cancer patients[J]. J Interferon Cytokine Res,2005,25(3):144-151.
11. Verdijk R, Franke HR, Wolbers F, et al. Differential effects of bisphosphonates on breast cancer cell lines[J]. Cancer Lett,2007,246(1-2):308-312.
12. Neville-webbe HL, Evans CA, Coleman RE, et al. Mechanisms of the synergistic interaction between the bisphosphonate zoledronic acid and the chemotherapy agent paclitaxel in breast cancer cells in vitro[J]. Tumor Biol, 2006,27(2):92-103.
13. Ottewell PD, Monkkonen H, Jones M, et al. Antitumor effects of doxorubicin followed by zoledronic acid in a mouse model of breast cancer[J]. J Natl Cancer Inst,2008,100(16):1167-1178.
14. Neville-webbe HL, Rostami-hojegan A, Evans CA, et al. Sequence-and schedule-dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells[J]. Int J Cancer,2005,113(3): 364-371.
15. Ottewell PD, Deux B, Monkkonen H, et al. Differential effect of doxorubicin and zoledronic acid on intraosseous versus extraosseous breast tumor growth in vivo[J]. Clin Cancer Res,2008,14(14):4658-4666.
16. Winter MC, Thorpe HC, Burkinshaw R, et al. Presentation title:The addition of zoledronic acid to neoadjuvant chemotherapy may influence pathological response exploratory evidence for direct anti-tumor activity in breast cancer. In:31th annual San Antonio breast cancer symposium,2008, abstract 5101.
17. Powles T, Paterson A, Mccloskey E, et al. Reduction in bone relapse and improved survival with oral clodronate for adjuvant treatment of operable breast cancer [ISRCTN83688026][J]. Breast Cancer Res,2006,8(2):R 13
18. Saarto T, Blpmqvist C, Virkkunen P, et al. Adjuvant clodronate treatment does not reduce the frequency of skeletal metastases in node-positive breast cancer patients:5-year results of a randomized controlled trial [J]. J Clin Oncol,2001, 19(1):10-17.
19. Saarto T, Vehmanen L, Blomqvist C, et al. Ten-year follow-up of a randomized controlled trial of adjuvant clodronate treatment in node-positive breast cancer patients[J]. J Clin Oncol.2004.22(14):9S-9S.
20. Crivellari D, Sun Z. Coates AS, et al. Letrozole compared with tamoxifen for elderly patients with endocrine-responsive early breast cancer:The BIG 1-98 trial[J]. J Clin Oncol,2008.26(12):1972-1979.
21. Coleman RE, Body JJ, Gralow JR, et al. Bone loss in patients with breast cancer receiving aromatase inhibitors and associated treatment strategies [J]. Cancer Treat Rev,2008,34:S31-S42.
22. Bundred NJ, Campbell ID, Davidson N, et al. Effective inhibition of aromatase inhibitor-associated bone loss by zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole[J]. Cancer,2008, 112(5):1001-1010.
23. Eidtmann H, Bundered NJ, Deboer R, et al. Presentation title:The Effect of Zoledronic Acid on Aromatase Inhibitor-Associated Bone Loss in Postmenopausal Women With Early Breast Cancer Receiving Adjuvant Letrozole:36 Months Follow-Up of ZO-FAST. SAN ANTONIO,2008: abstract 44.
24. Gnant M, Mlineritsch B, Luschin-Ebengreuth G, et al. Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer:5-year follow-up of the ABCSG-12 bone-mineral density substudy[J]. Lancet Oncol,2008,9(9):840-849.
25. Gnant M, Mlineritsch B, Schippinger W, et al. Adjuvant ovarian suppression combined with tamoxifen or anastrozole, alone or in combination with zoledronic acid, in premenopausal women with hormone-responsive, stage I and II breast cancer:First effi cacy results from ABCSG-12[J]. J Clin Oncol, ASCO Annual Meeting Proceedings 2008.26:abstract LBA4.
26. Bilezikian JP. Osteonecrosis of the jaw-Do biphosphonates pose a risk?[J]. N Engl J Med,2006,355(22):2278-2281.
1. Hopper-Borge E, Xu X, Shen T, et al. Human multidrug resistance protein 7 (ABCC10) is a resistance factor for nucleoside analogues and epothilone B[J]. Cancer Res,2009,69(1):178-184.
2. Galletti E, Magnani M. Renzulli ML. et al. Paclitaxel and docetaxel resistance: molecular mechanisms and development of new generation taxanes[J]. ChemMedChem,2007,2(7):920-942.
3. Rottenberg S, Nygren AO, Pajic M, et al. Selective induction of chemotherapy resistance of mammary tumors in a conditional mouse model for hereditary breast cancer[J]. Proc Natl Acad Sci USA,2007,104(29):12117-12122.
4. Maeno K, Ito K, Hama Y, et al. Mutation of the class Ⅰ beta-tubulin gene does not predict response to paclitaxel for breast cancer[J]. Cancer Lett,2003, 198(1):89-97.
5. Kamath K, Wilson L, Cabral F, et al. BetaⅢ-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability [J]. J Biol Chem,2005,280(13):12902-12907.
6. Tommasi S, Mangia A, Lacalamita R, et al. Cytoskeleton and paclitaxel sensitivity in breast cancer:the role of beta-tubulins[J]. Int J Cancer,2007, 120(10):2078-2085.
7. Rouzier R, Rajan R, Wagner P, et al. Microtubule-associated protein tau:a marker of paclitaxel sensitivity in breast cancer[J]. Proc Natl Acad Sci USA, 2005,102(23):8315-8320.
8. Andre F, Hatzis C, Anderson K, et al. Microtubule-associated protein-tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in estrogen receptor-positive breast cancer[J].Clin Cancer Res,2007,13(7): 2061-2067.
9. Gril B, Vidal M, Assayag F, et al. Grb2-SH3 ligand inhibits the growth of HER2+cancer cells and has antitumor effects in human cancer xenografts alone and in combination with docetaxel[J]. Int J Cancer,2007,121(2): 407-415.
10. Konecny GE, Thomssen C, Luck HJ, et al. Her-2/neu gene amplification and response to paclitaxel in patients with metastatic breast cancer[J]. J Natl Cancer Inst,2004,96(15):1141-1151.
11. Chen SC, Chang HK, Lin YC, et al. High pathologic complete response in HER 2-positive locally advanced breast cancer after primary systemic chemotherapy with weekly docetaxel and epirubicin[J]. Jpn J Clin Oncol, 2008,38(2):99-105.
12. Andre F, Mazouni C, Liedtke C, et al. HER2 expression and efficacy of preoperative paclitaxel/FAC chemotherapy in breast cancer[J]. Breast Cancer Res Treat,2008,108(2):183-190.
13. Estevez LG, Cuevas JM, Anton A, et al. Weekly docetaxel as neoadjuvant chemotherapy for stage Ⅱ and Ⅲ breast cancer:efficacy and correlation with biological markers in a phase Ⅱ. multicenter study[J]. Clin Cancer Res,2003, 9(2):686-692.
14. Tham YL, Gomez LF, Mohsin S, et al. Clinical response to neoadjuvant docetaxel predicts improved outcome in patients with large locally advanced breast cancers[J]. Breast Cancer Res Treat,2005,94(3):279-284.
15. Kim SJ, Miyoshi Y, Taguchi T, et al. High thioredoxin expression is associated with resistance to docetaxel in primary breast cancer[J]. Clin Cancer Res, 2005,11(23):8425-8430.
16. Nakayama S, Torikoshi Y, Takahashi T, et al. Prediction of paclitaxel sensitivity by CDK1 and CDK2 activity in human breast cancer cells[J]. Breast Cancer Res,2009,11(1):R12.
17. Vredenburg MR, Ojima I, Veith J, et al. Effects of orally active taxanes on P-glycoprotein modulation and colon and breast carcinoma drug resistance[J]. J Natl Cancer Inst,2001,93(16):1234-1245.
18. Patil Y, Sadhukha T, Ma L, et al. Nanoparticle-mediated simultaneous and targeted delivery of paclitaxel and tariquidar overcomes tumor drug resistance[J]. J Control Release,2009,136(1):21-29.
19. Mi Y and Lou L ZD6474 reverses multidrug resistance by directly inhibiting the function of P-glycoprotein[J]. Br J Cancer,2007,97(7):934-940.
20. Ferlini C, Raspaglio G, Mozzetti S, et al. The seco-taxane IDN5390 is able to target class III beta-tubulin and to overcome paclitaxel resistance[J].Cancer Res,2005,65(6):2397-2405.
21. Kim SN, Kim NH, Park YS, et al.7-Diethylamino-3(2'-benzoxazolyl) coumarin is a novel microtubule inhibitor with antimitotic activity in multidrug resistant cancer cells[J]. Biochem Pharmacol,2009,77(12): 1773-1779.
22. Pegram MD, Konecny GE, O'Callaghan C, et al. Rational combinations of trastuzumab with chemotherapeutic drugs used in the treatment of breast cancer[J]. J Natl Cancer Inst,2004,96(10):739-749.
23. Crown J, O'Leary M, and Ooi WS Docetaxel and paclitaxel in the treatment of breast cancer:a review of clinical experience [J]. Oncologist,2004,9 (Suppl 2):24-32.
24. Ting HJ, Hsu J, Bao BY, et al. Docetaxel-induced growth inhibition and apoptosis in androgen independent prostate cancer cells are enhanced by 1alpha,25-dihydroxyvitamin D3[J]. Cancer Lett,2007,247(1):122-129.
25. Marshall J, Chen H, Yang D, et al. A phase Ⅰ trial of a Bcl-2 antisense (G3139) and weekly docetaxel in patients with advanced breast cancer and other solid tumors[J]. Ann Oncol,2004,15(8):1274-1283.