酪氨酸激酶抑制剂tyrphostin AG1478对乳腺癌细胞作用的机制探讨

目的:探讨表皮生长因子受体(epithelial growth factor receptor,EGFR)的小分子酪氨酸激酶抑制剂tyrphostin AG1478对乳腺癌细胞的作用机制.方法:以乳腺癌细胞MCF-7(EGFR-/+)和MDA-MB-468(EGFR+++)为研究对象,采用MTT及克隆形成实验检测AG1478对乳腺癌MCF-7和MDA-MB-468细胞增殖和生长效应的影响;Western 印迹法测定EGFR、Akt和丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)的表达及活化水平;进一步应用FCM检测AG1478对MDA-MB-468细胞早期凋亡的影响.结果:AG1478 可抑制MDA-MB-468细胞的增殖和生长(P＜0.05),对MCF-7细胞增殖则无明显的抑制效应(P＞0.05);同时,MDA-MB-468细胞经AG1478处理后,细胞中磷酸化Akt(p-Akt)和磷酸化MAPK(p-MAPK)的表达水平明显降低(P＜0.05);FCM检测结果表明,AG1478可增加MDA-MB-468细胞的早期凋亡水平(P＜0.05).结论:AG1478可下调PI3K/Akt及Ras/Raf/MAPK信号通路,从而抑制乳腺癌MDA-MB-468细胞增殖及生长,并促进细胞凋亡;但其对MCF-7细胞无明显作用.     

EGF对ER阴性乳腺癌细胞株MAPK通路及c-fos的活化作用

目的比较表皮生长因子（EGF）对ER（＋）和ER（-）乳腺癌细胞MAPK信号转导通路及原癌基因c-fos表达的活化作用,探讨ER（-）的乳腺癌细胞激素非依赖性生长的可能机制.方法 EGF和MAPK抑制剂（PD98059）分别和（或）联合作用于ER（＋）乳腺癌细胞株MCF-7和ER（-）乳腺癌细胞株MDA-MB-435s,Western blot技术检测两种细胞中磷酸化MAPK蛋白（p-MAPK）及c-fos蛋白的表达.结果 EGF对ER阳性和阴性的两种细胞株p-MAPK和c-fos蛋白的表达均有上调作用,但MDA-MB-435s细胞中两种蛋白的激活明显高于MCF-7细胞,且c-fos蛋白表达与p-MAPK蛋白表达一致.结论 EGF通过激活MAPK信号转导通路,活化原癌基因c-fos来刺激细胞恶性增殖可能是引起ER（-）乳腺癌细胞的激素非依赖性生长的重要机制之一.     

雌激素受体β及其剪切变异体与乳腺癌他莫昔芬耐药的关系

目的探讨雌激素受体（ER）亚型（ERβ）及其剪切变异体（ERβcx）的表达与乳腺癌他莫昔芬耐药的关系。方法 （1）对乳腺癌他莫西芬敏感MCF-7细胞和他莫西芬耐药MCF-7细胞进行培养,利用去甲基化物5-氮杂胞苷（5-Aza-2-deoxycytidine,5-AZA-CdR）去甲基化的作用,对MCF-7细胞进行药物处理获得MCF-75-AZA-CdR细胞,采用Western blot方法检测3组细胞中ERβ和ERβcx蛋白的表达。（2）取对数生长期生长良好的乳腺癌MCF-7细胞株,按2.5×103细胞/孔接种于96孔细胞培养板板中,实验组分为他莫细芬处理的MCF-7细胞（MCF-7＋TAM组）和MCF-75-AZA-CdR细胞（MCF-75-AZA-CdR细胞＋TAM组）,对照组为MCF-7细胞,采用MTT比色法,观察3组细胞的增殖情况。统计学分析采用单因素方差分析和重复测量方差分析。结果 （1）ERβ蛋白在他莫西芬敏感MCF-7细胞中的表达高于他莫西芬耐药的MCF-7细胞,两组细胞间差异有显著的统计学意义（P=0.000）;ERβcx蛋白表达在两组之间差异无统计学意义（P=0.366）。MCF-75-AZA-CdR细胞ERβ和ERβcx蛋白表达均高于他莫西芬敏感MCF-7细胞和他莫西芬敏耐药MCF-7细胞（P均=0.000）。（2）与对照组MCF-7细胞相比,他莫西芬明显降低了MCF-7＋TAM组和MCF-75-AZA-CdR细胞＋TAM组的细胞增值速度并抑制细胞生长;且他莫西芬抑制细胞增殖作用MCF-75-AZA-CdR细胞＋TAM组强于MCF-7＋TAM组（P=0.000）。结论 ERβ蛋白在他莫西芬敏感MCF-7细胞中的表达高。MCF-75-AZA-CdR细胞中ERβ和ERβcx蛋白的表达均高。他莫西芬抑制细胞增殖作用在他莫西芬处理的MCF-75-AZA-CdR细胞中强     

雌激素受体β在乳腺癌他莫昔芬内分泌治疗耐药中的作用

目的：探讨雌激素受体β（estrogen receptor β,ERβ）的表达与乳腺癌他莫昔芬（tamoxifen,TAM）内分泌治疗耐药的相关性及其机制。方法：以前期构建的ERα/ERβ不同表达的人乳腺癌MCF-7细胞株\[M/HK（阴性对照）、M/siα（ERαlow/ERβhigh）、M/siβ（ERαhigh/ERβlow）细胞\]为研究对象,MTT法评估乳腺癌细胞对TAM的耐药性;用半定量RT-PCR法检测细胞中耐药相关基因MDR1、TOPOⅡ、LRP和GST-π的mRNA表达水平,用Western blotting法检测细胞中耐药相关信号通路MAPK、PI3K/Akt的p-ERK、p-Akt蛋白表达水平。结果：与对照组MCF-7细胞相比,MCF-7细胞中的ERβ高表达可促进高浓度TAM（1、5、10 μmol/L）对MCF-7细胞增殖的抑制作用\[（45.788 ± 1.641）% vs （24.288±1.170）%,（57.899±1.583）% vs（31.499±1.978）%,（59.853 ±1.648）% vs（38.039±1.482）%;均P<0.05 ）\],该抑制作用与TAM浓度呈剂量依赖性。ERβ高表达可显著抑制MCF-7细胞耐药基因MDR1、TOPOⅡ、LRP的 mRNA表达水平（0.431±0.032 vs 0.932±0.083,0.234±0.008 vs 0.391±0.002,0.47±0.028 vs 0.586±0.036;均P<0.05);可显著下调Akt和ERK蛋白的磷酸化水平（0224±0.006 vs 0.437±0.007,0.367±0.015 vs 0.756±0.039;均P<0.05)。结论： ERβ表达水平可影响乳腺癌细胞MCF-7对TAM的耐药性,该作用机制可能与耐药基因的表达及PI3K/ AKT、MAPK信号通路激活有关。     

依维莫司与PI3K抑制剂联合他莫昔芬对ER阳性乳腺癌细胞系作用的观察

目的:探讨他莫昔芬(TAM)联合mTORC抑制剂依维莫司(EVE)和PI3K抑制剂LY294002(LY)阻断负反馈激活,分析对ER(+)乳腺癌细胞系增殖、凋亡和周期的影响。方法:乳腺癌细胞系MCF-7和BT474分为对照组、TAM、TAM+EVE、TAM+LY和TAM+EVE+LY 5组,分别检测肿瘤细胞的增殖、凋亡与周期以及信号通路的改变情况。结果:MCF-7和BT474接受药物处理以后,EVE明显抑制肿瘤细胞的增殖,A450分别为0.23和0.32,特别是在三药联合组,抑制细胞增殖更加显著,A450分别为0.16和0.20,P<0.05。细胞凋亡的研究显示,EVE可促进肿瘤细胞凋亡,三药联合以后,乳腺癌细胞MCF-7和BT474的凋亡率分别为30.1%和54.2%。细胞周期的研究显示,G1期比例升高,三药联合后MCF-7和BT474G1期比率分别为86.02%和84.91%。EVE可有效阻断mTOR信号通路的活化,但通过负反馈作用可导致磷酸化AKT 473位点的激活,而联合PI3K抑制剂后,这种负反馈作用被阻断。结论:内分泌治疗药物TAM联合mTOR抑制剂EVE和PI3K抑制剂可抑制ER(+)乳腺癌细胞增殖、促进细胞凋亡、阻滞细胞生长,达到更好的治疗效果。     

维甲酸联合他莫昔芬对人乳腺癌细胞株作用的研究

目的:探讨他莫昔芬(TAM)与维甲酸(ATRA)联合对人乳腺癌他莫昔芬耐药株的作用.方法:在体外培养条件下,分别或联合应用ATRA和TAM作用于MCF-7人乳腺癌他莫昔芬耐药株(MCF-7/T)及敏感组(MCF-7/S).MTT比色法分析细胞生长抑制作用;用流式细胞仪(FCM)检测细胞周期分布、凋亡率及用药前后Bcl-2、Bax、Fas和FasL蛋白的变化.结果:TAM能抑制ER阳性MCF-7/S的生长,阻滞细胞周期于G0/G1期,并可诱导细胞凋亡,TAM不能抑制MCF-7/T的生长; ATRA预处理细胞24 h后,TAM抗乳腺癌细胞MCF-7/S的作用增强,且恢复了MCF-7/T对TAM的敏感性.ATRA与TAM联用后,MCF-7/T细胞Bcl-2蛋白表达下调,细胞Bax、Fas和FasL蛋白表达水平未见明显变化.结论:体外条件下,TAM通过影响细胞周期、诱导细胞凋亡而发挥抗ER阳性MCF-7/S作用;视黄酸能加强TAM对激素敏感细胞MCF-7/S的抗乳腺癌作用,恢复耐受细胞MCF-7/T对TAM的敏感性.同时恢复TAM对MCF-7/T的促凋亡作用.     

获得性他莫昔芬耐受MCF-7细胞膜受体的变化及格列卫的逆转作用

目的：探讨耐受他莫昔芬(TAM)的MCF-7细胞膜受体等的变化及格列卫对耐药的逆转作用。方法：MTY法检测TAM对E2、EGF、Insulin刺激的MCF-7增殖的抑制作用；流式细胞术检测与10^-7mol／LTAM孵育8周、16周后的MCF-7细胞ER、PR、EGFR及VEGF的变化，MTY法检测格列卫对耐药的逆转作用。结果：TAM抑制E2、EGF、Insulin刺激的MCF-7增殖；耐受TAM的MCF-7ER、PR表达率由父代的93％、91％下降为41％、36％，EGFR由10％上升为39％，VEGF由36％下降至30％，对EGF刺激的增殖更敏感，格列卫可增强TAM的生长抑制作用。结论：耐受TAM的MCF-7细胞ER、PR表达下降，EGFR表达升高，EGF／EGFR刺激的细胞增殖作用增强，格列卫可能作为对TAM获得性耐药后逆转用药方案或贯序用药方案。     

miR-181a表达水平与ER阳性乳腺癌对内分泌治疗敏感性的关系

目的:研究miR-181a表达水平与雌激素受体（estrogen receptor,ER）阳性乳腺癌对内分泌治疗敏感性之间的关系。方法:采用梯度递增法诱导乳腺癌他莫昔芬耐药细胞株,利用集落形成实验检测细胞对他莫昔芬的敏感性。利用RT-PCR法检测miR-181a表达的情况,利用KM-plotter分析miR-181表达与乳腺癌患者预后之间的关系。结果:我们成功的诱导了MCF-7他莫昔芬继发耐药细胞株TAM-R。RT-PCR分析结果显示:他莫昔芬继发耐药的TAM-R细胞株及原发他莫昔芬耐药BT474细胞株中miR-181a的表达水平均显著高于对他莫昔芬敏感的MCF-7细胞株。生存分析结果显示:miR-181a的表达水平与ER阴性乳腺癌患者的总生存无关,但在ER阳性乳腺癌中miR-181a表达高的患者总生存明显差于miR-181a低表达者。进一步分析结果显示:在ER阳性但未接受内分泌治疗的乳腺癌患者中miR-181a表达与总生存无关,但在ER阳性且接受过内分泌治疗的乳腺癌患者中miR-181a高表达者总生存显著差于miR-181a低表达者。结论:miR-181a高表达ER阳性乳腺癌患者对内分泌治疗敏感性差,miR-181a可能是预测ER 阳性乳腺癌对内分泌治疗敏感性的指标。     

GPR30在乳腺癌内分泌治疗耐受中的作用研究

G蛋白偶联受体-30(G protein-coupled receptor 30,GPR30)是一种新型膜性结合性雌激素受体(estrogen receptor,ER).他莫昔芬(tamoxifen,TAM)及其代谢产物44}他莫昔芬(4-hydroxtamoxifen,OHT)是GPR30激动剂.GPR30的激活可能导致TAM耐药性的发生,GPR30可反式激活表皮生长因子受体(epidermal growth factor receptor,EGFR),介导E2增殖效应,可能与ERα具有协同作用,参与乳腺癌细胞增殖、侵袭和转移等行为.TAM作为ERα(+)的竞争性抑制剂,在乳腺癌内分泌治疗中占据着重要的地位,但是长期应用TAM的耐药限制了其临床应用.EGFR及ER相关信号途径,在TAM耐药机制中扮演重要角色.研究证实GPR30可反转TAM变成促生长激动剂,促进肿瘤生长.本文从GPR30的作用机制和TAM的耐药性机制联系为出发点,探讨GPR30在TAM耐药性中的作用.     

人参皂甙延缓乳腺癌三苯氧胺耐药的研究

目的研究人参皂苷Rg3延缓乳腺癌互苯氧胺（TAM）耐药的作用,并探究其最佳作用浓度。方法TAM诱导野生型MCF-7乳腺癌细胞构建TAM耐药细胞系TAM—R,将WT—MCF-7乳腺癌细胞与TAM—R细胞分别培养于不同浓度TAM培养液中,MTT法比较细胞生长变化。WT—MCF-7分别培养于含不同浓度Rg3的TAM培养液中,MTF法检测细胞对TAM的反应。结果经TAM处理后,TAM—R细胞生长曲线明显高于wⅢr—MCF一7细胞的生长曲线,人参皂甙Rg3呵延缓WT—MCF-7细胞TAM耐药的发生时间,1×10-8μmol／L和5×10-8μmol／L人参皂甙R妇延缓耐药时间最长。结论临床可通过联合应用参一胶囊和TAM延缓内分泌耐药。     

Inhibition of phosphoinositide 3-kinase enhances the cytotoxicity of AG1478, an epidermal growth factor receptor inhibitor, in breast cancer cells

Aberrant activation and dysfunction of the EGFR/PI3K/Akt signaling pathways are commonly reported in breast cancer. Constitutive activation of the PI3K/Akt pathway by the lack of PTEN regulation is associated with resistance to novel targeted therapies including EGFR inhibitors. We aimed to study whether Ly294002, an inhibitor of PI3K, could enhance the cytotoxicity of AG1478, an inhibitor of EGFR, on breast cancer cells. We tested these agents in the MDA-MB-468 and MCF-7 breast cancer cell lines with different EGFR and PTEN profiles (MDA-MB-468: high expression of EGFR and PTEN mutation; MCF-7: low expression of EGFR and PTEN wild type). Simultaneous inhibition of EGFR and PI3K in MDA-MB-468 cells with combined Ly294002 and AG1478 treatment had a greater anti-proliferative effect and increased mitotic death than either treatment alone. In addition, more apoptosis and increased induction of cell arrest at G0/G1 phase were observed in MDA-MB-468 cells with the combined treatment. Phosphor-EGFR and its downstream signal transducer, phosphor-Akt, were fully attenuated only by simultaneous treatment with Ly294002 and AG1478. These data suggest that the inhibition of PI3K could enhance the cytotoxicity of EGFR inhibitors on breast cancer cells and tumors which overexpress EGFR and demonstrate mutated PTEN. This dual inhibition treatment protocol may have important therapeutic implication in the treatment of a subset of breast cancer patients with high expression of EGFR and deficient function of PTEN.     

Role of PKC-ERK signaling in tamoxifen-induced apoptosis and tamoxifen resistance in human breast cancer cells

This study was designed to investigate the role of protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in tamoxifen (TAM)-induced apoptosis and drug resistance in human breast cancer cells. Drug-sensitive, or estrogen receptor (ER)-positive human breast carcinoma cells (MCF-7) and the multi-drug-resistant variant (ER-negative) MCF-7/ADR cells were treated with doses of TAM for various periods of time. Cell viability and apoptosis were assessed using cell counting, DNA fragmentation and flow cytometric analysis. We found that TAM administration caused a significant increase in apoptosis of MCF-7 cells but not MCF-7/ADR cells. Western blot analysis revealed enhanced expression of PKCδ but decreased expression of PKCα in ER-positive MCF-7 cells; while ER-negative MCF-7/ADR cells had decreased levels of PKCδ and increased levels of PKCα. Interestingly, we observed that in MCF-7 cells, TAM stimulated apoptosis by promoting rapid activation of PKCδ, antagonizing downstream signaling of ERK phosphorylation; while in MCF-7/ADR cells, TAM upregulated PKCα, which promoted ERK phosphorylation. These results suggest that PKCδ enhances apoptosis in TAM-treated MCF-7 cells by antagonizing ERK phosphorylation; while the PKCα pathway plays an important role in TAM-induced drug resistance by activating ERK signaling in MCF-7/ADR cells. The combination of TAM with PKCα and ERK inhibitors could promote TAM-induced apoptosis in breast cancer cells.     

Role of GPR30 in the mechanisms of tamoxifen resistance in breast cancer MCF-7 cells

Tamoxifen is the most frequently used anti-hormonal drug for treatment of women with hormone-dependent breast cancer. The aim of this study is to investigate the mechanism of tamoxifen resistance and the impact of the new estrogen G-protein coupled receptor (GPR30). MCF-7 cells were continuously exposed to tamoxifen for 6 months to induce resistance to the inhibitory effect of tamoxifen. These tamoxifen-resistant cells (TAM-R) exhibited enhanced sensitivity to 17-ß-estradiol and GPR30 agonist, G1, when compared to the parental cells. In TAM-R cells, tamoxifen was able to stimulate the cell growth and MAPK phosphorylation. These effects were abolished by EGFR inhibitor AG1478, GPR30 anti-sense oligonucleotide, and the selective c-Src inhibitor PP2. Only EGFR basal expression was slightly elevated in the TAM-R cells, whereas GPR30 expression and the basal phosphorylation of Akt and MAPK remained unchanged when compared to the parental cells. Interestingly, estrogen treatment significantly increased GPR30 translocation to the cell surface, which was stronger in TAM-R cells. Continuous treatment of MCF-7 cells with GPR30 agonist G1 mimics the long-term treatment with tamoxifen and increases drastically its agonistic activity. This data suggests the important role of GPR30/EGFR receptor signaling in the development of tamoxifen resistance. The inhibition of this pathway is a valid option to improve anti-hormone response in breast cancer.     

Estrogen receptor-α36 is involved in development of acquired tamoxifen resistance via regulating the growth status switch in breast cancer cells

Acquired tamoxifen (TAM) resistance limits the therapeutic benefit of TAM in patients with hormone-dependent breast cancer. The switch from estrogen-dependent to growth factor-dependent growth is a critical step in this process. However, the molecular mechanisms underlying this switch remain poorly understood. In this study, we established a TAM resistant cell sub line (MCF-7/TAM) from estrogen receptor-α (ER-α66) positive breast cancer MCF-7 cells by culturing ER-α66-positive MCF-7 cells in medium plus 1 μM TAM over 6 months. MCF-7/TAM cells were then found to exhibit accelerated proliferation rate together with enhanced in vitro migratory and invasive ability. And the estrogen receptor-α36 (ER-α36), a novel 36-kDa variant of ER-α66, was dramatically overexpressed in this in vitro model, compared to the parental MCF-7 cells. Meanwhile, the expression of epidermal growth factor receptor (EGFR) in MCF-7/TAM cells was significantly up-regulated both in mRNA level and protein level, and the expression of ER-α66 was greatly down-regulated oppositely. In the subsequent studies, we overexpressed ER-α36 in MCF-7 cells by stable transfection and found that ER-α36 transfected MCF-7 cells (MCF-7/ER-α36) similarly exhibited decreased sensitivity to TAM, accelerated proliferative rate and enhanced in vitro migratory and invasive ability, compared to empty vector transfected MCF-7 cells (MCF-7/V). Real-time qPCR and Western blotting analysis revealed that MCF-7/ER-α36 cells possessed increased EGFR expression but decreased ER-α66 expression both in mRNA level and protein level, compared to MCF-7/V cells. This change in MCF-7/ER-α36 cells could be reversed by neutralizing anti-ER-α36 antibody treatment. Furthermore, knock-down of ER-α36 expression in MCF-7/TAM cells resulted in reduced proliferation rate together with decreased in vitro migratory and invasive ability. Decreased EGFR mRNA and protein expression as well as increased ER-α66 mRNA expression were also observed in MCF-7/TAM cells with down-regulated ER-α36 expression. In addition, blocking EGFR/ERK signaling in MCF-7/ER-α36 cells could restore the expression of ER-α66 partly, suggesting a regulatory function of EGFR/ERK signaling in down-regulation of ER-α66 expression. In conclusion, our results indicated for the first time a regulatory role of ER-α36 in up-regulation of EGFR expression and down-regulation of ER-α66 expression, which could be an underlying mechanism for the growth status switch in breast tumors that contribute to the generation of acquired TAM resistance. And ER-α36 could be considered a potential new therapeutic target in breast tumors which have acquired resistance to TAM.     

Estrogen receptor α is the major driving factor for growth in tamoxifen-resistant breast cancer and supported by HER/ERK signaling

Resistance to tamoxifen is a major clinical challenge in the treatment of breast cancer; however, it is still unclear which signaling pathways are the major drivers of tamoxifen-resistant growth. To characterize resistance mechanisms, we have generated different tamoxifen-resistant breast cancer cell lines from MCF-7. In this model, we investigated whether signaling from human epidermal growth factor receptors (HERs), their downstream kinases, or from the estrogen receptor α (ERα) was driving tamoxifen-resistant cell growth. Increased expression of EGFR and increased phosphorylation of HER3 were observed upon acquisition of tamoxifen resistance, and the extracellular activated kinase (ERK) signaling pathway was highly activated in the resistant cells. The EGFR inhibitor gefitinib and the ERK pathway inhibitor U0126 resulted in partial and preferential growth inhibition of tamoxifen-resistant cells. All the tamoxifen-resistant cell lines retained ERα expression but at a lower level compared to that in MCF-7. Importantly, we showed via ERα knockdown that the tamoxifen-resistant cells were dependent on functional ERα for growth and we observed a clear growth stimulation of resistant cell lines with clinically relevant concentrations of tamoxifen and 4-OH-tamoxifen, indicating that tamoxifen-resistant cells utilize agonistic ERα stimulation by tamoxifen for growth. The tamoxifen-resistant cells displayed high phosphorylation of ERα at Ser118 in the presence of tamoxifen; however, treatment with U0126 neither affected the level of Ser118 phosphorylation nor expression of the ERα target Bcl-2, suggesting that ERK contributes to cell growth independently of ERα in our cell model. In support of this, combined treatment against ERα and ERK signaling in resistant cells was superior to single-agent treatment and as effective as fulvestrant treatment of MCF-7 cells. Together, these findings demonstrate that ERα is a major driver of growth in tamoxifen-resistant cells supported by HER/ERK growth signaling, implying that combined targeting of these pathways may have a clinical potential for overcoming tamoxifen resistance.     

Silibinin suppresses EGFR ligand-induced CD44 expression through inhibition of EGFR activity in breast cancer cells

CD44, the transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. The expression of CD44 and its variants is associated with poor prognosis in breast cancer. Here, we investigated the effect of silibinin (a polyphenolic flavonolignan of the herbal plant of Silybum marianum, milk thistle) on the epidermal growth factor (EGF) ligand-induced CD44 expression in human breast cancer cells. The levels of CD44 mRNA and protein expression were greatly increased by EGF and by TGF-α in SKBR3 and BT474 breast cancer cells. In contrast, EGFR ligand-induced CD44 expression was reduced by EGFR inhibitors, AG1478 and lapatinib, respectively. Interestingly, we observed that EGFR ligand-induced CD44 and matrix metalloproteinase-9 (MMP-9) expression was reduced by silibinin treatment in a dose-dependent manner. In addition, silibinin suppressed the EGF-induced phosphorylation of EGFR and extracellular signal-regulated kinase1/2 (ERK1/2), a downstream signaling molecule of EGFR. Therefore, we suggest that silibinin prevents the EGFR signaling pathway and may be used as an effective drug for the inhibition of metastasis of human breast cancer.     

ADAM12 induces estrogen-independence in breast cancer cells

Antiestrogen therapy has been used successfully to prolong disease-free and overall survival of ER positive breast cancer patients. However, 50% of patients with ER+ tumors fail to respond to such therapy or eventually acquire resistance to endocrine therapy, resulting in tumor progression and mortality. It is imperative, therefore, to understand the mechanisms that lead to hormone refractory breast cancer in order to develop therapeutics that can modulate the resistance to antiestrogen therapy. The protease, ADAM12, can be detected in the urine of breast cancer patients and its levels correlate with disease status, stage, and cancer risk. Within the context of this study, the authors have investigated the role of the two distinct isoforms of ADAM12 in breast tumor cell proliferation and as potential mediators of endocrine resistance. Using stable clones of ADAM12-overexpressing MCF-7 cells, the authors analyzed proliferation rates of these ER+ breast tumor cells both in estrogen-depleted medium and in the presence of the antiestrogens, tamoxifen, and ICI 182,780. Acquired estrogen resistance in these cells was analyzed using phospho-RTK analysis. Upregulation and phosphorylation of proteins were detected via immunoprecipitation and immunoblotting. EGFR and MAPK inhibitors were used to explore the mechanism of acquired estrogen resistance in breast tumor cells. It was observed that overexpression of the two isoforms, transmembrane ADAM12-L, and secreted ADAM12-S, in breast tumor cells promoted estrogen-independent proliferation. In ADAM12-L-expressing cells, estrogen-independence was a direct result of increased EGFR expression and MAPK activation, whereas, the mechanism in ADAM12-S-expressing cells may be enhanced IGF-1R signaling. The importance of the EGFR signaling pathway in the estrogen-independent growth of ADAM12-L expressing cells was highlighted by the effect of EGFR inhibitors AG1478 and PD15035 or MAPK inhibitor U0126, each of which abolished the antiestrogen resistance in these cells. Taken together, these results demonstrate that ADAM12 isoforms confer a proliferative advantage to MCF-7 cells in the absence of estrogen stimulation, and suggest that downregulation of ADAM12 in combination with endocrine therapy may represent a useful pharmacological approach to breast cancer therapy.