新型雌激素受体亚型ERα36在乳腺癌中作用的初步研究
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摘要
乳腺癌是女性最为常见的恶性肿瘤之一。美国癌症协会研究表明,美国每年新增乳腺癌病例约194280例,为女性发病率最高的恶性肿瘤,因乳腺癌死亡的病例达40610例,死亡率仅次于肺癌而居第二位。随着我国经济社会的不断发展,近来年我国乳腺癌发病率也呈现明显上升趋势,已位居女性恶性肿瘤发病率的首位。因此,如何有效对乳腺癌进行三级预防,提高综合治疗的效果,降低发病率与死亡率,已成为肿瘤学当前的研究重点之一
目前乳腺癌的综合治疗包括外科手术切除,药物治疗以及局部放射治疗等手段。其中以三苯氧胺和芳香化酶抑制剂为代表的内分泌治疗(Endocrine therapy)因其具有确切的疗效和良好的耐受性,在乳腺癌的药物治疗中占有重要地位。而肿瘤组织中雌激素受体(Estrogen receptor, ER)的状态被广泛认为是预测患者对内分泌治疗反应性和预后的关键指标之一。目前一般认为,雌激素受体有两个亚型,即ERα和ERβ。其中分子量为66kD的ERa (ERa66)首先于1986年被克隆,属配体活化的核转录因子,具有六个功能区(A-F),构成数个结构域。其中N端的A／B区构成转录活化结构域AF-1,介导非配体依赖的转录活化功能。C区负责结合特定的DNA序列及形成受体二聚体,D区为铰链区。C端的E／F区构成另一个结构域AF-2,为配体依赖的转录活化结构域。
近年来研究发现,由于剪切异构体(Splice variants)的存在,ERα尚有数个亚型。Wang等于2005年发现了一个分子量为36kD的新型ERα亚型,命名为ERα36。与全长ERα66相比,该亚型缺少转录活化结构域AF-1及AF-2,但仍保留DNA结合域及部分二聚体形成域和配体结合域,并在C端具有一个由27个氨基酸所组成的新型结构域。前期研究表明,ERα36在乳腺癌细胞系及乳腺癌组织中均有表达,且通过在人胚胎肾细胞系HEK293中的研究表明,由于缺乏内在的转录活性,ERα36可竞争性抑制ERα66所介导的受体依赖或非依赖的转录活化,但可介导MAPK/ERK信号通路的活化。目前尚未有文献系统报道ERα36在人乳腺癌发生发展中所起的作用。我们的前期工作表明,ERα36在结直肠癌中的表达低于临近正常组织,在结直肠癌的发生发展中起一定的作用。为了研究ERα36与人乳腺癌的关系,本课题检测了ERα36在74对乳腺癌及其相应临近正常乳腺组织中的表达水平；构建了ERα36的真核表达载体,并在低表达ERα36的人乳腺癌细胞系MCF-7中得到稳定表达；研究了ERα36转染前后乳腺癌细胞系MCF-7对雌激素、三苯氧胺的反应性以及对化疗药物紫杉醇敏感性的差异。具体内容如下：
第一部分：ERα36和ERα66在乳腺癌及临近正常乳腺组织中的表达。采用实时定量PCR技术,检测ERα36和ERα66的mRNA在74对乳腺癌及其相应临近正常乳腺组织中表达的差异,分析其在乳腺癌发生过程中可能的作用。同时,结合上述病例的临床病理资料,如年龄、绝经状态、肿瘤大小、淋巴结受累情况及肿瘤分期等,分析乳腺癌组织中ERα36和ERα66的表达与上述指标之间的关系。结果表明,乳腺癌组织中ERα36的表达水平显著低于相应临近正常乳腺组织,且ERα36的低表达与较大的肿瘤直径、淋巴结受累及较高的肿瘤分期有关,提示ERα36表达降低与乳腺癌的发生过程及进展转移过程密切相关。我国女性乳腺癌及临近正常乳腺组织ERα66的表达规律与美国非洲裔女性乳腺癌相仿,提示二者具有共同的临床特征,可按照相似的策略进行内分泌治疗。
第二部分：ERα36真核表达载体的构建表达及其对人乳腺癌MCF-7细胞系生物学行为的影响。本课题组的前期工作已克隆并鉴定了ERα36的全长编码序列并成功连接于真核表达载体pcDNA3.1(+)上,构建出ERα36的真核表达载体pcDNA3.1(+)-ERa36.由于尚未有商业化的特异性ERα36抗体,给鉴定细胞内的ERα36表达情况带来了很大的困难,因此,本课题将FLAG标签肽编码序列片段插入pcDNA3.1(+)-ERa36载体中,成功构建pcDN A3.1 (+)-FLAG-ERa36真核表达体系,并经测序确认。将上述ERα36表达载体稳定转染人乳腺癌细胞系MCF-7,经Westernblot鉴定该蛋白在MCF-7细胞中表达。应用MTT法检测转染ERα36前后,人乳腺癌细胞系MCF-7对雌激素、三苯氧胺的反应性以及对化疗药物紫杉醇敏感性的变化。结果表明,在雌激素的作用下,转染ERα36的MCF-7细胞的增殖能力明显高于转染空载体的对照细胞,而在三苯氧胺的作用下,转染ERα36的MCF-7细胞的增殖能力也明显提高。通过对治疗乳腺癌常用化疗药物紫杉醇的敏感性试验表明,转染ERα36的MCF-7细胞对紫杉醇的细胞毒作用更为敏感,生长抑制作用更明显。研究结果提示,肿瘤组织高表达ERα36的乳腺癌患者接受三苯氧胺治疗带来的获益更小,选择ERα阳性乳腺癌患者进行三苯氧胺治疗需考虑肿瘤组织中ERα36的表达状况。同时,对于肿瘤组织高表达ERα36的患者,选择紫杉醇化疗可能带来更大的生存获益。
Breast cancer is the most commonly diagnosed cancer type in women and ranks the second in female cancer deaths. American Cancer Sociaty estimates that about 194,280 new occurrences and 40,610 deaths are expected each year in the United States. The incidence rate of Chinese female breast cancer is also increasing. In certain industrialized cities, breast cancer has ranked first in female cancer incidences. Therefore, more understandings about breast cancer are needed for the improvement of early detection and comprehensive treatment.
While surgery, chemotherapy, and radiotherapy have become established modalities for breast cancer treatment, endocrine therapy, such as tamoxifen and aromatase inhibitors, can provide further survival benefits with well tolerances. Among all molecular factors involved in breast cancer management, the expression status of estrogen receptor (ER) has been widely accepted as a prognostic marker and a predictor for endocrine therapy response. There are two subtypes of ER, ERa and ERβ.The 66kD ERα(here also termed ERα66), first cloned and sequenced in 1986, consists of six functional regions, from the N-terminal domain A/B to the C-terminal domain F. The N-terminal domain A/B is responsible for the ligand-independent transactivation function (AF-1). Domain C is responsible for receptor dimerization and DNA binding. Domain D is hinge region. The C-terminal domain E/F is responsible for the ligand-dependent transactivation (AF-2). Several ERβdomains show a high degree of homology to ERa, but its exact functions in breast cancer are still unclear.
A single ERa66 was considered to be responsible for all ERa related estrogen biological actions in breast cancer until the discovery of its splice variants. These variants have been identified in both breast cancer cell lines. The novel 36kD variant (here termed ERa36) which was first identified and cloned in 2005 by Wang et al., lacks both transcriptional activation function domains (AF-1 and AF-2) but retains the DNA binding domain, partial dimerization, and ligand-binding domains campared to the full-length ERa66. Previous studies have confirmed ERa36 expression in breast cancer tissues and breast cancer cell lines. In human embryonic kidney cell line HEK293, it was found that ERa36 may influence ERa66 mediated transcriptional activity, but can induce the activation of the mitogenactivated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathway. Our earlier studies showed a decreased ERa36 mRNA level in colorectal cancers compared to their matched normal tissues. However, no critical study results on the effects of ERa36 in breast cancer were reported. In the present study, we compared the expression of ERa36 and ERa66 mRNA in 74 paired breast cancers and normal breast tissues, and investigated its function on cell growth and response to estrogen, tamoxifen and paclitaxel by transfecting ERa36 into ERa36-low-expression MCF-7 breast cancer cells. The details are listed below. Part One:The expression of ERa36 and ERa66 mRNA level in breast cancers and their matched normal tissues.
Real-time quantitative PCR assay was applied to quantify the mRNA levels of ERa36 and ERa66 in 74 pairs of breast cancers and their matched normal tissues. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control for normalization. Besides, the findings were correlated with the clinicopathological characteristics of these breast cancer patients, including age, menopausal status, tumor size, lymph node metastasis, and tumor stage. Quantitative analysis showed that the ERa36 mRNA levels in both ERa positive and negative breast cancers were significantly lower than those in their matched normal tissues (P<0.001). The ERa36 mRNA level in breast cancers over 2 cm was significantly lower compared with cancers less than 2 cm (P=0.014). Similarly, cancers with lymph node metastasis showed significantly decreased ERa36 mRNA levels than those without lymph nodes involvement (P=0.023). The ERa36 mRNA level was also decreased in advanced diseases compared to early stage cancers (P=0.031). These results all suggest that ERa36 may associate with breast carcinogenesis and progression. The expression pattern of ERa66 in Chinese female breast cancer patients showed a close resemblance to those from African-American and Taiwanese women. Part Two:Construction and expression of ERa36 eukaryotic expression system in breast cancer cell line MCF-7 and its effects on response to estrogen, tamoxifen and paclitaxel.
Our previous work had finished cloning of the full length coding region of ERa36 and had ligated it with the eukaryotic expression vector pcDNA3.1(+). However, since there is no commercial antibody against ERa36 available currently, it was not possible to confirm the successful transfection of ERa36 into breast cancer cell lines. Therefore, the coding sequence of FLAG tag was cloned and inserted into the expression vector to construct the eukaryotic expression system pcDNA3.1 (+)-FLAG-ERα36, which was verified by sequencing later. The expression vector was successfully transfected into MCF-7 breast cancer cells and the expression of ERa36 protein was confirmed by Western blot.
MTT assay was performed to evaluate the influence of ERa36 on the response to estrogen, tamoxifen and paclitaxel in MCF-7 cells. At the presence of estradiol, the growth rate of ERa36 transfected MCF-7 cells was higher than those control cells which transfected with empty vectors. Suprisingly, when incubated with tamoxifen, same growth acceleration of ERa36 transfected MCF-7 cells was observed. Besides, ERa36 transfected MCF-7 cells tended to be more sensitive to paclitaxel, which showed more significant growth suppression compared to those control cells. These findings suggest that patients express relative high level of ERa36 in breast cancers are less likely to benefit from tamoxifen therapy. However, these patients are the right population for paclitaxel treatment.
目前乳腺癌的综合治疗包括外科手术切除,药物治疗以及局部放射治疗等手段。其中以三苯氧胺和芳香化酶抑制剂为代表的内分泌治疗(Endocrine therapy)因其具有确切的疗效和良好的耐受性,在乳腺癌的药物治疗中占有重要地位。而肿瘤组织中雌激素受体(Estrogen receptor, ER)的状态被广泛认为是预测患者对内分泌治疗反应性和预后的关键指标之一。目前一般认为,雌激素受体有两个亚型,即ERα和ERβ。其中分子量为66kD的ERa (ERa66)首先于1986年被克隆,属配体活化的核转录因子,具有六个功能区(A-F),构成数个结构域。其中N端的A／B区构成转录活化结构域AF-1,介导非配体依赖的转录活化功能。C区负责结合特定的DNA序列及形成受体二聚体,D区为铰链区。C端的E／F区构成另一个结构域AF-2,为配体依赖的转录活化结构域。
近年来研究发现,由于剪切异构体(Splice variants)的存在,ERα尚有数个亚型。Wang等于2005年发现了一个分子量为36kD的新型ERα亚型,命名为ERα36。与全长ERα66相比,该亚型缺少转录活化结构域AF-1及AF-2,但仍保留DNA结合域及部分二聚体形成域和配体结合域,并在C端具有一个由27个氨基酸所组成的新型结构域。前期研究表明,ERα36在乳腺癌细胞系及乳腺癌组织中均有表达,且通过在人胚胎肾细胞系HEK293中的研究表明,由于缺乏内在的转录活性,ERα36可竞争性抑制ERα66所介导的受体依赖或非依赖的转录活化,但可介导MAPK/ERK信号通路的活化。目前尚未有文献系统报道ERα36在人乳腺癌发生发展中所起的作用。我们的前期工作表明,ERα36在结直肠癌中的表达低于临近正常组织,在结直肠癌的发生发展中起一定的作用。为了研究ERα36与人乳腺癌的关系,本课题检测了ERα36在74对乳腺癌及其相应临近正常乳腺组织中的表达水平；构建了ERα36的真核表达载体,并在低表达ERα36的人乳腺癌细胞系MCF-7中得到稳定表达；研究了ERα36转染前后乳腺癌细胞系MCF-7对雌激素、三苯氧胺的反应性以及对化疗药物紫杉醇敏感性的差异。具体内容如下：
第一部分：ERα36和ERα66在乳腺癌及临近正常乳腺组织中的表达。采用实时定量PCR技术,检测ERα36和ERα66的mRNA在74对乳腺癌及其相应临近正常乳腺组织中表达的差异,分析其在乳腺癌发生过程中可能的作用。同时,结合上述病例的临床病理资料,如年龄、绝经状态、肿瘤大小、淋巴结受累情况及肿瘤分期等,分析乳腺癌组织中ERα36和ERα66的表达与上述指标之间的关系。结果表明,乳腺癌组织中ERα36的表达水平显著低于相应临近正常乳腺组织,且ERα36的低表达与较大的肿瘤直径、淋巴结受累及较高的肿瘤分期有关,提示ERα36表达降低与乳腺癌的发生过程及进展转移过程密切相关。我国女性乳腺癌及临近正常乳腺组织ERα66的表达规律与美国非洲裔女性乳腺癌相仿,提示二者具有共同的临床特征,可按照相似的策略进行内分泌治疗。
第二部分：ERα36真核表达载体的构建表达及其对人乳腺癌MCF-7细胞系生物学行为的影响。本课题组的前期工作已克隆并鉴定了ERα36的全长编码序列并成功连接于真核表达载体pcDNA3.1(+)上,构建出ERα36的真核表达载体pcDNA3.1(+)-ERa36.由于尚未有商业化的特异性ERα36抗体,给鉴定细胞内的ERα36表达情况带来了很大的困难,因此,本课题将FLAG标签肽编码序列片段插入pcDNA3.1(+)-ERa36载体中,成功构建pcDN A3.1 (+)-FLAG-ERa36真核表达体系,并经测序确认。将上述ERα36表达载体稳定转染人乳腺癌细胞系MCF-7,经Westernblot鉴定该蛋白在MCF-7细胞中表达。应用MTT法检测转染ERα36前后,人乳腺癌细胞系MCF-7对雌激素、三苯氧胺的反应性以及对化疗药物紫杉醇敏感性的变化。结果表明,在雌激素的作用下,转染ERα36的MCF-7细胞的增殖能力明显高于转染空载体的对照细胞,而在三苯氧胺的作用下,转染ERα36的MCF-7细胞的增殖能力也明显提高。通过对治疗乳腺癌常用化疗药物紫杉醇的敏感性试验表明,转染ERα36的MCF-7细胞对紫杉醇的细胞毒作用更为敏感,生长抑制作用更明显。研究结果提示,肿瘤组织高表达ERα36的乳腺癌患者接受三苯氧胺治疗带来的获益更小,选择ERα阳性乳腺癌患者进行三苯氧胺治疗需考虑肿瘤组织中ERα36的表达状况。同时,对于肿瘤组织高表达ERα36的患者,选择紫杉醇化疗可能带来更大的生存获益。
Breast cancer is the most commonly diagnosed cancer type in women and ranks the second in female cancer deaths. American Cancer Sociaty estimates that about 194,280 new occurrences and 40,610 deaths are expected each year in the United States. The incidence rate of Chinese female breast cancer is also increasing. In certain industrialized cities, breast cancer has ranked first in female cancer incidences. Therefore, more understandings about breast cancer are needed for the improvement of early detection and comprehensive treatment.
While surgery, chemotherapy, and radiotherapy have become established modalities for breast cancer treatment, endocrine therapy, such as tamoxifen and aromatase inhibitors, can provide further survival benefits with well tolerances. Among all molecular factors involved in breast cancer management, the expression status of estrogen receptor (ER) has been widely accepted as a prognostic marker and a predictor for endocrine therapy response. There are two subtypes of ER, ERa and ERβ.The 66kD ERα(here also termed ERα66), first cloned and sequenced in 1986, consists of six functional regions, from the N-terminal domain A/B to the C-terminal domain F. The N-terminal domain A/B is responsible for the ligand-independent transactivation function (AF-1). Domain C is responsible for receptor dimerization and DNA binding. Domain D is hinge region. The C-terminal domain E/F is responsible for the ligand-dependent transactivation (AF-2). Several ERβdomains show a high degree of homology to ERa, but its exact functions in breast cancer are still unclear.
A single ERa66 was considered to be responsible for all ERa related estrogen biological actions in breast cancer until the discovery of its splice variants. These variants have been identified in both breast cancer cell lines. The novel 36kD variant (here termed ERa36) which was first identified and cloned in 2005 by Wang et al., lacks both transcriptional activation function domains (AF-1 and AF-2) but retains the DNA binding domain, partial dimerization, and ligand-binding domains campared to the full-length ERa66. Previous studies have confirmed ERa36 expression in breast cancer tissues and breast cancer cell lines. In human embryonic kidney cell line HEK293, it was found that ERa36 may influence ERa66 mediated transcriptional activity, but can induce the activation of the mitogenactivated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathway. Our earlier studies showed a decreased ERa36 mRNA level in colorectal cancers compared to their matched normal tissues. However, no critical study results on the effects of ERa36 in breast cancer were reported. In the present study, we compared the expression of ERa36 and ERa66 mRNA in 74 paired breast cancers and normal breast tissues, and investigated its function on cell growth and response to estrogen, tamoxifen and paclitaxel by transfecting ERa36 into ERa36-low-expression MCF-7 breast cancer cells. The details are listed below. Part One:The expression of ERa36 and ERa66 mRNA level in breast cancers and their matched normal tissues.
Real-time quantitative PCR assay was applied to quantify the mRNA levels of ERa36 and ERa66 in 74 pairs of breast cancers and their matched normal tissues. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an endogenous control for normalization. Besides, the findings were correlated with the clinicopathological characteristics of these breast cancer patients, including age, menopausal status, tumor size, lymph node metastasis, and tumor stage. Quantitative analysis showed that the ERa36 mRNA levels in both ERa positive and negative breast cancers were significantly lower than those in their matched normal tissues (P<0.001). The ERa36 mRNA level in breast cancers over 2 cm was significantly lower compared with cancers less than 2 cm (P=0.014). Similarly, cancers with lymph node metastasis showed significantly decreased ERa36 mRNA levels than those without lymph nodes involvement (P=0.023). The ERa36 mRNA level was also decreased in advanced diseases compared to early stage cancers (P=0.031). These results all suggest that ERa36 may associate with breast carcinogenesis and progression. The expression pattern of ERa66 in Chinese female breast cancer patients showed a close resemblance to those from African-American and Taiwanese women. Part Two:Construction and expression of ERa36 eukaryotic expression system in breast cancer cell line MCF-7 and its effects on response to estrogen, tamoxifen and paclitaxel.
Our previous work had finished cloning of the full length coding region of ERa36 and had ligated it with the eukaryotic expression vector pcDNA3.1(+). However, since there is no commercial antibody against ERa36 available currently, it was not possible to confirm the successful transfection of ERa36 into breast cancer cell lines. Therefore, the coding sequence of FLAG tag was cloned and inserted into the expression vector to construct the eukaryotic expression system pcDNA3.1 (+)-FLAG-ERα36, which was verified by sequencing later. The expression vector was successfully transfected into MCF-7 breast cancer cells and the expression of ERa36 protein was confirmed by Western blot.
MTT assay was performed to evaluate the influence of ERa36 on the response to estrogen, tamoxifen and paclitaxel in MCF-7 cells. At the presence of estradiol, the growth rate of ERa36 transfected MCF-7 cells was higher than those control cells which transfected with empty vectors. Suprisingly, when incubated with tamoxifen, same growth acceleration of ERa36 transfected MCF-7 cells was observed. Besides, ERa36 transfected MCF-7 cells tended to be more sensitive to paclitaxel, which showed more significant growth suppression compared to those control cells. These findings suggest that patients express relative high level of ERa36 in breast cancers are less likely to benefit from tamoxifen therapy. However, these patients are the right population for paclitaxel treatment.
引文
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