Androgens act synergistically to enhance estrogen-induced upregulation of human tissue kallikreins 10, 11, and 14 in breast cancer cells via a membrane bound androgen receptor

The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well as in the pathogenesis of endocrine-related cancers, especially breast cancer. However, clinical data suggest that combined testosterone and estrogen treatments on post-menopausal women increase the risk of breast cancer. Experiments have shown that many, if not all kallikreins are under steroid hormone regulation in breast cancer cell lines. Their implication as prognostic and diagnostic markers has also been well-documented. Thus, we investigated the effect of combined hormone stimulation with androgens and 17β-estradiol on the ductal caricinoma cell line BT474. This cell line has been shown to be sensitive to both, androgens (secreting PSA) and estrogens (secreting a number of kallikreins including KLK10, 11, and KLK14). We found that PSA expression was downregulated upon combined hormone stimulation, confirming reports that estrogen can antagonize and block the activity of the androgen receptor. Upon analysis of estrogen-sensitive kallikreins 10, 11, and 14, all showed to be synergistically enhanced in their expression three- to fourfold, upon joint hormone treatment versus individual hormone stimulation. The enhancement is dependent upon the action of androgens as treatment with the androgen receptor antagonist cyproterone actetate normalized the expression of KLK10, 11, and KLK14 to estrogen-stimulation levels. The synergistic effects between estrogens and androgens on estrogen-sensitive genes may have implications on the role of the kallikreins in associated risk of breast cancer and progression.     

Significance of steroid sulfatase expression in human breast cancer

The sulfatase pathway has been thought to be a primary means of local production of estrone in human breast cancer tissue. We measured steroid sulfatase (STS) mRNA levels in 97 breast cancers and evaluated its association with disease-free survival. High levels of STS mRNA proved to be a significant predictor of reduced relapse-free survival, both as a continuous variable (log STS mRNA; P=0.028) and as a dichotomous variable with an optimized cutoff point (P=0.002). In multivariate analysis a high level of STS mRNA was an independent factor for predicting relapse-free survival. These results suggest a putative role of STS in breast cancer growth and metastasis, and administration of sulfatase inhibitors to breast cancer patients with high levels of STS mRNA might be an additional treatment option.     

Efficacy of three potent steroid sulfatase inhibitors: pre-clinical investigations for their use in the treatment of hormone-dependent breast cancer

Estrogenic steroids, such as estradiol, are known to play a crucial role in the development and growth of hormone-dependent breast cancer. Steroid sulfatase (STS) inhibitors that can prevent the biosynthesis of these steroids via the sulfatase pathway offer therapeutic potential. We show here the in vivo profile, including the efficacy in a xenograft breast cancer model and pharmacokinetics, of three potent STS inhibitors. MCF-7 cells stably over-expressing STS cDNA (MCF-7STS) were generated. Ovariectomised, MF-1, female nude mice receiving subcutaneous injections of estradiol sulfate (E2S) and bearing MCF-7STS xenografts, were orally treated with the STS inhibitors STX64, STX213, and STX1938. Treatment was administered once weekly at a dose of 1 mg/kg for 35 days during which animals received E2S thrice weekly. Mice were weighed and tumor measurements taken weekly. Furthermore, the pharmacokinetics for STX213 was determined in rats. STX213 and STX1938 exhibited potent STS inhibition in vivo. However, STX1938 demonstrated a greater duration of activity. In vehicle treated nude mice receiving E2S, tumor volumes increased by 260% after 35 days compared to day zero. STX64 (1 mg/kg) failed to reduce tumor growth when given once weekly. STX213 and STX1938 (once weekly, 1 mg/kg) significantly inhibited (P < 0.05) tumor growth over this same time period. These compounds completely inhibited liver and tumor STS activity and significantly reduced the levels of plasma E2. This study indicates that the STS inhibitor, STX213, exhibits excellent efficacy and pharmacokinetics and therefore offers a potentially novel treatment for hormone-dependent breast cancer.     

乳腺癌C—erbB—2和nm23基因表达的研究

目的：探讨Ｃ－ｅｒｂＢ－２和ｎｍ２３基因表达与乳腺癌组织学类型、临床分期以及对乳腺癌淋巴结转移的影响。方法：９８例乳腺癌术后石腊包埋标本行免疫组化ＳＰ法。结果：Ｃ－ｅｒｂＢ－２在淋巴结转移组表达率为６０．２９％,在非淋巴结转移组表达率为３３．３３％,差别有显著意义;Ｃ－ｅｒｂＢ－２在乳腺癌不同临床分期表达率各不相同,Ⅳ期为１１．７８％,Ⅱｂ＋Ⅲ期为５６．６０％,Ⅰ＋Ⅱａ期为２５．９２％,差别有显著     

乳腺癌基因表达谱研究进展

乳腺癌是分子表达差异显著的一组疾病,乳腺癌临床异质性源自分子异质性.根据基因表达谱研究开发的分子标签,如70-基因标签、21-基因标签、乳腺癌分子分型标签等,在预测乳腺癌患者预后、指导临床治疗决策方面,表现出良好的应用价值和前景.     

Predicting features of breast cancer with gene expression patterns

Data from gene expression arrays hold an enormous amount of biological information. We sought to determine if global gene expression in primary breast cancers contained information about biologic, histologic, and anatomic features of the disease in individual patients. Microarray data from the tumors of 129 patients were analyzed for the ability to predict biomarkers [estrogen receptor (ER) and HER2], histologic features [grade and lymphatic-vascular invasion (LVI)], and stage parameters (tumor size and lymph node metastasis). Multiple statistical predictors were used and the prediction accuracy was determined by cross-validation error rate; multidimensional scaling (MDS) allowed visualization of the predicted states under study. Models built from gene expression data accurately predict ER and HER2 status, and divide tumor grade into high-grade and low-grade clusters; intermediate-grade tumors are not a unique group. In contrast, gene expression data is inaccurate at predicting tumor size, lymph node status or LVI. The best model for prediction of nodal status included tumor size, LVI status and pathologically defined tumor subtype (based on combinations of ER, HER2, and grade); the addition of microarray-based prediction to this model failed to improve the prediction accuracy. Global gene expression supports a binary division of ER, HER2, and grade, clearly separating tumors into two categories; intermediate values for these bio-indicators do not define intermediate tumor subsets. Results are consistent with a model of regional metastasis that depends on inherent biologic differences in metastatic propensity between breast cancer subtypes, upon which time and chance then operate. Xuesong Lu and Xin Lu contributed equally to this work.     

A novel steroidal selective steroid sulfatase inhibitor KW-2581 inhibits sulfated-estrogen dependent growth of breast cancer cells in vitro and in animal models

We screened a series of 17β-(N-alkylcarbamoyl)-estra-1,3,5(10)trine-3-O-sulfamate derivatives, and describe here a potent and selective steroid sulfatase (STS) inhibitor with antitumor effects in breast cancer models in vitro and in vivo. In biochemical assays using crude enzymes isolated from recombinant Chinese hamster ovary cells expressing human arylsulfatses (ARSs), one of the best compounds, KW-2581, inhibited STS activity with an IC₅₀ of 4.0 nM, while > 1000-fold higher concentrations were required to inhibit the other ARSs. The failure to stimulate the growth of MCF-7 human breast cancer cells as well as in uteri in ovariectomized rats indicated the lack of estrogenicity of this compound. In MCF-7 cells transfected with the STS gene, termed MCS-2 cells, KW-2581 inhibited the growth of cells stimulated by estrone sulfate (E1S) but also 5-androstene-3β, 17β-diol 3-sulfate (ADIOLS) and dehydroepiandrostenedione 3-sulfate. We found that oral administration of KW-2581 inhibited both E1S- and ADIOLS-stimulated growth of MCS-2 cells in a mouse hollow fiber model. In a nitrosomethylurea-induced rat mammary tumor model, KW-2581 induced regression of E1S-stimulated tumor growth as effectively as tamoxifen or another STS inhibitor, 667 Coumate. Dose-response studies in the same rat model demonstrated that more than 90% inhibition of STS activity in tumors was necessary to induce tumor shrinkage. STS activity in tumors has well correlated with that in leukocytes, suggesting that STS activity in leukocytes could be used as an easily detectable pharmacodynamic marker. These findings demonstrate that KW-2581 is a candidate for development as a therapeutic agent for the treatment of hormone receptors-positive breast cancer.     

Inhibition of steroid sulfatase activity and cell proliferation in ZR-75-1 and BT-474 human breast cancer cells by KW-2581 in vitro and in vivo

In the present study, we found that two hormone receptor-positive human breast cancer cell lines, ZR-75-1 and BT-474, naturally expressed steroid sulfatase (STS) protein and had catalytic activity to produce estrone from estrone sulfate (E1S) with a comparable level to those in human breast cancer tissues. E1S at physiological concentrations stimulated the growth of those cells. A novel steroidal STS inhibitor, KW-2581 inhibited the STS activity of ZR-75-1 cells with an IC₅₀ of 13 nM, a potency equal to or higher than that of the non-steroidal STS inhibitor, 667 COUMATE. The inhibitory effect of KW-2581 was enhanced by pre-incubation with STS enzyme, suggests being irreversible inhibition. KW-2581 inhibited the E1S-stimulated growth of ZR-75-1 cells with an IC₅₀ of 0.18 nM, but failed to inhibit the growth stimulated by 17β-estradiol. Expression of E1S-induced progesterone receptors in ZR-75-1 cells was reduced by treatment of KW-2581 at concentrations as low as 0.1 nM. Oral administration of KW-2581 for 4 weeks caused tumor shrinkage in a mouse xenograft model. Tumor STS activity had been completely (>95%) eliminated by 24 hours after the last administration. These findings suggest that KW-2581 has considerable potential for therapeutic development as a novel anti-hormonal drug for treatment of breast cancer.     

Human UDP-Glucuronosyltransferases: Effects of altered expression in breast and pancreatic cancer cell lines

Increased aerobic glycolysis and de novo lipid biosynthesis are common characteristics of invasive cancers. UDP-glucuronosyltransferases (UGTs) are phase II drug metabolizing enzymes that in normal cells possess the ability to glucuronidate these lipids and speed their excretion; however, de-regulation of these enzymes in cancer cells can lead to an accumulation of bioactive lipids, which further fuels cancer progression. We hypothesize that UGT2B isoform expression is down-regulated in cancer cells and that exogenous re-introduction of these enzymes will reduce lipid content, change the cellular phenotype, and inhibit cancer cell proliferation. In this study, steady-state mRNA levels of UGT isoforms from the 2B family were measured using qPCR in 4 breast cancer and 5 pancreatic cancer cell lines. Expression plasmids for UGT2B isoforms known to glucuronidate cellular lipids, UGT2B4, 2B7, and 2B15 were transfected into MCF-7 and Panc-1 cells, and the cytotoxic effects of these enzymes were analyzed using trypan blue exclusion, annexin V/PI staining, TUNEL assays, and caspase-3 immunohistochemistry. There was a significant decrease in cell proliferation and a significant increase in the number of dead cells after transfection with each of the 3 UGT isoforms in both cell lines. Cellular lipids were also found to be significantly decreased after transfection. The results presented here support our hypothesis and emphasize the important role UGTs can play in cellular proliferation and lipid homeostasis. Evaluating the effect of UGT expression on the lipid levels in cancer cell lines can be relevant to understanding the complex regulation of cancer cells, identifying the roles of UGTs as “lipid-controllers” in cellular homeostasis, and illustrating their suitability as targets for future clinical therapy development.     

Gene expression profiling of breast cancer

Numerous genes are controlled by complex regulatory networks and involved in the development and progression of breast cancer, and these genes are the key factors determining each characteristic of the tumor. Gene expression profiling, a large scale analysis of gene expression, has created new possibilities for the molecular characterization of cancer. Systematic analysis of expression patterns of thousands of genes in tumor cells using DNA microarrays and correlation of these patterns to specific features of phenotypic variation may provide the basis for an improved taxonomy of cancer. These profiles have the potential to explain the genetic heterogeneity of breast cancer and allow treatment strategies to be planned in accordance with their probability of success in individual patients.     

重组人GM-CSF逆转录病毒基因转移系统的建立及其在人肿瘤细胞中的稳定表达

目的　建立逆转录病毒介导的 GM- CSF基因转移系统 ,为 GM- CSF基因修饰的肿瘤细胞疫苗研究奠定实验基础。方法　应用基因重组技术将 GM- CSF c DNA克隆于逆转录病毒载体p DORneo,获得重组载体 p DORGM。经脂质体介导将其转染于病毒包装细胞系 PA317细胞 ,经NIH3T3细胞检测病毒滴度 ,NIH3T3放大实验检测辅助病毒 ,并用高滴度病毒感染人乳腺癌细胞系MCF- 7。结果　GM- CSF基因克隆入逆转录病毒载体 ,经 PA317细胞包装产生病毒 ,最高的病毒滴度为 1× 10 6CFU/ ml,且没有发现辅助病毒存在。重组病毒感染的人乳腺癌 MCF- 7细胞在体外长期培养中保持稳定分泌 GM- CSF,活性在 50 0～ 80 0 U/ 10 6细胞 / 2 4小时。结论　建立的逆转录病毒介导的GM- CSF基因转移系统安全、有效 ,为 GM- CSF应用于肿瘤基因治疗提供了实验基础。     

高通量基因检测系统MammaPrint与乳腺癌预后预测

化疗是乳腺癌患者术后最重要的辅助治疗手段.由于传统的临床病理指标不能准确预测患者复发风险,因此不能准确筛选出哪些患者需要化疗、哪些患者不需要化疗.高通量基因检测系统MammaPrint已被证实可较准确预测相当一部分乳腺癌患者复发风险,因而可用于指导个体化治疗.本文就MammaPrint的临床应用及研究现状作一综述.     

RGS5在乳腺癌组织和细胞系中的表达和定位

目的:探讨G蛋白信号转导调节蛋白5（RGS5）在人乳腺癌细胞系中的表达。方法:提取人正常乳腺上皮细胞HBL100,人乳腺癌细胞系MCF-7,ZR-75-1,ZR-75-30,MDA-MB-231,HCC1937的总蛋白和总RNA,提取细胞的总蛋白和总RNA进行蛋白免疫印迹和实时定量PCR检测RGS5的表达。共聚焦激光扫描显微镜观察RGS5胞内定位;免疫组化检测RGS5在乳腺肿瘤组织中表达情况。结果:MDA-MB-231细胞系中RGS5基因表达上调,MCF7和ZR-75-1细胞系中RGS5蛋白表达提高。结论:RGS5在乳腺癌细胞的高表达可能参与其生长和转移,揭示RGS5可能是人乳腺肿瘤的治疗靶点。     

乳腺癌纤溶分子标志物t-PA、u-PA表达的临床研究

目的:检测乳腺癌患者血浆纤溶分子标志物含量、mRNA水平的变化以探索其与肿瘤浸润、播散的关系及临床意义。方法:采用逆转录、实时定量PCR技术检测30例乳腺癌组织中组织型纤溶酶原激活剂(tPA)、尿激酶型纤溶酶原激活剂(uPA)mRNA水平;用ELISA法同步检测患者血浆纤溶分子标志物含量,包括tPA、uPA、尿激酶型纤溶酶原激活剂受体(uPAR)、纤溶酶抗纤溶酶复合物(PAP)等。结果:乳腺癌患者血浆uPA、uPAR、PAP含量均较正常对照明显升高,其中,有周围淋巴结转移、远处脏器转移者uPA升高更为显著。肿瘤细胞uPAmRNA表达增高,在不同临床分期组中有显著差异,而tPAmRNA则减少。雌孕激素受体双阳性组tPAmRNA表达较其他组为高。结论:乳腺癌患者纤溶功能明显亢进,可能是其易播散、浸润的主要原因之一。逆转录实时定量PCR技术,使tPAmRNA、uPAmRNA有望成为临床上乳腺癌病情监测、预后判断及治疗选择的辅助指标。     

类固醇硫酸酯酶在乳腺癌和正常乳腺组织中的表达及其意义

目的:探讨类固醇硫酸酯酶(steroid sulfatase,STS)蛋白及其mRNA在乳腺癌组织及相对应的正常乳腺组织中的表达,以及与临床病理特征之间的关系.方法:分别应用免疫组织化学SP法和RT-PCR法检测40例乳腺癌患者乳腺癌组织及其对应的癌旁正常乳腺组织中STS蛋白和mRNA的表达水平,并进行比较,同时分析STS蛋白表达与临床病理特征之间的关系.结果: STS蛋白主要表达于乳腺癌细胞和正常乳腺组织上皮细胞的细胞质内,有3例患者乳腺癌细胞的细胞核中可见STS蛋白的表达,乳腺间质组织未见STS蛋白表达.乳腺癌组织及正常乳腺组织中STS蛋白的表达水平与其mRNA的表达水平均呈正相关.乳腺癌组织中STS蛋白的阳性表达率(70.0%)明显高于其对应的正常乳腺组织(42.5%),差异有统计学意义(P=0.013).分层分析显示,绝经前、淋巴结转移和病理分期较晚的乳腺癌患者,其乳腺癌组织中STS蛋白的阳性表达率明显高于其对应的正常乳腺组织(P<0.05).结论:乳腺癌组织可能通过高表达STS以促进自身合成雌激素,提高肿瘤局部的雌激素水平,从而促进肿瘤的生长和转移.同时,随着肿瘤的生长,肿瘤局部合成的雌激素在肿瘤生长中的作用可能越来越重要.     

非小细胞肺癌细胞株抗癌药物敏感性相关基因差异表达的初步研究

背景与目的为了提高晚期肺癌的化疗效果，实行个体化治疗，筛选和鉴定肺癌细胞抗癌药物敏感性基因具有重要临床意义。本研究比较了非小细胞肺癌(NSCLC)和永生化人支气管上皮细胞(BET2A)细胞株抗癌药物敏感性相关基因的差异表达，以寻找与抗癌药物敏感性相关的基因。方法应用cDNA macroarray技术，对6株NSCLC和BET2A细胞株的抗癌药物敏感性相关基因进行差异表达分析，RT-PCR技术验证滤膜杂交结果。结果在1291个候选基因中筛选出73个差异表达基因，其中45个基因表达上调，28个基因表达下调。RT-PCR验证结果与cDNA macroarray检测结果一致。结论抗癌药物敏感性相关基因的差异表达可能是药物敏感性产生不同的原因之一。本研究结果为逆转肺癌多药耐药性提供了理论基础和新靶点，并为临床新药开发及个体化治疗提供了实验依据。     

Comparison of gene expression profiles predicting progression in breast cancer patients treated with tamoxifen

Background Molecular signatures that predict outcome in tamoxifen treated breast cancer patients have been identified. For the first time, we compared these response profiles in an independent cohort of (neo)adjuvant systemic treatment naïve breast cancer patients treated with first-line tamoxifen for metastatic disease. Methods From a consecutive series of 246 estrogen receptor (ER) positive primary tumors, gene expression profiling was performed on available frozen tumors using 44K oligoarrays (n = 69). A 78-gene tamoxifen response profile (formerly consisting of 81 cDNA-clones), a 21-gene set (microarray-based Recurrence Score), as well as the HOXB13-IL17BR ratio (Two-Gene-Index, RT-PCR) were analyzed. Performance of signatures in relation to time to progression (TTP) was compared with standard immunohistochemical (IHC) markers: ER, progesterone receptor (PgR) and HER2. Results In univariate analyses, the 78-gene tamoxifen response profile, 21-gene set and HOXB13-IL17BR ratio were all significantly associated with TTP with hazard ratios of 2.2 (95% CI 1.3–3.7, P = 0.005), 2.3 (95% CI 1.3–4.0, P = 0.003) and 4.2 (95% CI 1.4–12.3, P = 0.009), respectively. The concordance among the three classifiers was relatively low, they classified only 45–61% of patients in the same category. In multivariate analyses, the association remained significant for the 78-gene profile and the 21-gene set after adjusting for ER and PgR. Conclusion The 78-gene tamoxifen response profile, the 21-gene set and the HOXB13-IL17BR ratio were all significantly associated with TTP in an independent patient series treated with tamoxifen. The addition of multigene assays to ER (IHC) improves the prediction of outcome in tamoxifen treated patients and deserves incorporation in future clinical studies.