Obesity Suppresses Estrogen Receptor Beta Expression in Breast Cancer Cells via a HER2-Mediated Pathway

Obesity is associated with a worse breast cancer prognosis, while greater breast tumor estrogen receptor beta (ERβ) expression is correlated with improved therapy response and survival. The objective of this study was to determine the impact of obesity on breast cancer cell ERβ expression, which is currently unknown. We utilized an in vitro model of obesity in which breast cancer cells were exposed to patient serum pooled by body mass index category (obese (OB): ≥30 kg/m²; normal weight (N): 18.5–24.9 kg/m²). Four human mammary tumor cell lines representing the major breast cancer subtypes (SKBR3, MCF-7, ZR75, MDA-MB-231) and mammary tumor cells from MMTV-neu mice were used. ERβ expression, assessed by qPCR and western blotting, was suppressed in the two HER2-overexpressing cell lines (SKBR3, MMTV-neu) following OB versus N sera exposure, but did not vary in the other cell lines. Expression of Bcl-2 and cyclin D1, two genes negatively regulated by ERβ, was elevated in SKBR3 cells following exposure to OB versus N sera, but this difference was eliminated when the ERβ gene was silenced with siRNA. Herceptin, a HER2 antagonist, and siRNA to HER2 were used to evaluate the role of HER2 in sera-induced ERβ modulation. SKBR3 cell treatment with OB sera plus Herceptin increased ERβ expression three-fold. Similar results were obtained when HER2 expression was silenced with siRNA. OB sera also promoted greater SKBR3 cell viability and growth, but this variance was not present when ERβ was silenced or the cells were modified to overexpress ERβ. Based on this data, we conclude that obesity-associated systemic factors suppress ERβ expression in breast cancer cells via a HER2-mediated pathway, leading to greater cell viability and growth. Elucidation of the mechanism(s) mediating this effect could provide important insights into how ERβ expression is regulated as well as how obesity promotes a more aggressive disease.     

HER2/ErbB2-induced Breast Cancer Cell Migration and Invasion Require p120 Catenin Activation of Rac1 and Cdc42

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.     

The Bacterial Protein Azurin Impairs Invasion and FAK/Src Signaling in P-Cadherin-Overexpressing Breast Cancer Cell Models

P-cadherin overexpression occurs in about 30% of all breast carcinomas, being a poor prognostic factor for breast cancer patients. In a cellular background of wild-type E-cadherin, we have previously shown that its expression promotes invasion, motility and migration of breast cancer cells due to the induced secretion of metalloproteases (MMPs) to the extracellular medium and to the concomitant shedding of a pro-invasive soluble form of this protein (sP-cad). Azurin is secreted by Pseudomonas aeruginosa and induces in vitro and in vivo cytotoxicity after its preferential penetration in human cancer cells relative to normal cells. Three different breast cancer cell lines, MCF-7/AZ.Mock, MCF-7/AZ.Pcad and SUM149 were treated with sub-killing doses of azurin. Invasion of these cells was measured using Matrigel Invasion Assays and MTT assays were performed to determine cell viability upon treatment and the effects on cadherins expression was determined by Western blot and Immunofluorescence. Gelatin Zymography was used to determine activity of MMP2 in the conditioned media of azurin treated and untreated cells and the phosphorylation levels of intracellular signaling proteins were determined by Western blot. The invasive phenotype of these breast cancer cells was significantly reduced by azurin. Azurin (50–100 µM) also caused a specific decrease on P-cadherin protein levels from 30–50% in MCF-7/AZ.Pcad and SUM149 breast cancer cell lines, but the levels of E-cadherin remain unaltered. More, the levels of sP-cad and the activity of MMP2 were reduced in the extracellular media of azurin treated cells and we also observed a decrease in the phosphorylation levels of both FAK and Src proteins. Our data show that azurin specifically targets P-cadherin, not E-cadherin, abrogating P-cadherin-mediated invasive effects and signaling. Therefore, azurin could possibly be considered a therapeutic tool to treat poor-prognosis breast carcinomas overexpressing P-cadherin in a wild type E-cadherin context.     

NUAK1通过影响F-actin聚合促进乳腺癌的侵袭转移

本课题组先前研究证明NUAK1/ARK5参与乳腺癌、胶质瘤侵袭转移,但机制尚不清楚.本文证明,NUAK1通过影响F-actin聚合促进乳腺癌的侵袭转移.应用小RNA干扰技术敲除乳腺癌细胞系MDA-MB-231中的NUAK1,用G418进行稳定筛选,并用Western印迹进行蛋白质表达检测,结果显示,成功敲除MDA-MB-231细胞中的NUAK1;采用Transwell侵袭实验检测NUAK1在乳腺癌细胞侵袭转移中的作用,结果表明,NUAK1被干扰的SiNUAK1/MDA-231细胞的侵袭能力明显减弱;应用免疫荧光法对细胞的F-actin进行荧光染色,半定量F-actin聚合分析结果显示,IGF-1在转染空载的细胞组（Scr/MDA-231）能诱导肌动蛋白短暂的聚合反应,而在敲除NUAK1的细胞组（SiNUAK1/MDA-231）肌动蛋白的聚合显著减少;用细胞因子IGF-1刺激乳腺癌细胞观察cofilin磷酸化,结果显示,在敲除NUAK1的细胞组（SiNUAK1/MDA-231）,IGF-1诱导的cofilin的磷酸化明显受抑制.上述结果表明,NUAK1能通过促进F-actin的聚合从而影响乳腺癌细胞的侵袭转移.     

Oleanolic Acid Suppresses Migration and Invasion of Malignant Glioma Cells by Inactivating MAPK/ERK Signaling Pathway

Mitogen-activated protein kinases/Extracellular signal-regulated kinase (MAPK/ERK) pathway is essential for migration and invasion of malignant glioma. It is efficient to inhibit migration and invasion of glioma cells by targeting this pathway. Oleanolic acid (OA) has been well demonstrated to suppress survival, growth and angiogenesis of glioma cells. However, it is still unknown if OA affects the migration and invasion of glioma cells. We utilized U-87 MG glioma cell lines and primary glioma cells from patients to study the effect of OA on migration and invasion of glioma cells with multidisciplinary approaches. In this study, we found that OA significantly decreased the ability of glioma cells to migrate and invade. Epithelial-mesenchymal transition (EMT) of glioma cells was also suppressed by OA treatment. Furthermore, MAPK/ERK pathway was greatly inhibited in glioma cells under OA treatment. MAPK/ERK reactivation induced by a recombinant lentiviral vector, Lv-MEK, was able to rescue the inhibitory effect of OA on migration and invasion of glioma cells. Taken together, we provided evidences that OA was a MAPK/ERK pathway-targeting anti-tumor agent. Although the concentrations we used exceeded its physiological level, OA may be used to prevent migration and invasion of glioma cells by developing its derivatives with enhanced bioactivity.     

整合素β2促进人乳腺癌细胞MCF-7的迁移,侵袭与粘附

本研究主要目标为探讨整合素β2 (ITGB2)的高表达对人乳腺癌细胞MCF-7迁移,侵袭与粘附能力的影响。本研究首先构建了ITGB2过表达质粒,实验设阴性对照组(pcDNA-3.1+)与ITGB2基因过表达组(pcDNA-3.1+/ITGB2)。ITGB2过表达质粒转染MCF-7细胞后,采用逆转录PCR与Western blotting方法分别检测ITGB2 mRNA转录水平与蛋白翻译水平;流式细胞术检测细胞周期的改变;划痕实验检测细胞横向迁移能力;Transwell小室实验检测细胞纵向迁移能力及侵袭能力;人脐静脉血管内皮细胞(HUVEC)粘附实验检测癌症细胞与血管内皮细胞之间的粘附能力,Western blotting实验检测侵袭相关指标MMP9,整合素经典通路中FAK蛋白磷酸化水平的改变。研究结果表明:转染ITGB2过表达质粒后,MCF-7细胞中ITGB2的m RNA水平(p<0.01)与蛋白水平(p<0.05)均显著增高;流式细胞术实验中,实验组S期的细胞所占比例与对照组无明显差异;划痕实验与Transwell小室实验中,实验组的迁移侵袭能力显著性增强;人脐静脉血管内皮细胞粘附实验中,实验组乳腺癌细胞与血管内皮细胞的粘附能力强于对照组(p<0.05);且Western blotting结果显示MMP9和p-FAK蛋白水平明显上升。由以上结果可得出结论,过表达ITGB2后会增强人乳腺癌细胞MCF-7的迁移、侵袭与粘附能力,而对其增殖能力无明显影响。     

EFEMP2 Mediates GALNT14-Dependent Breast Cancer Cell Invasion

N-Acetylgalactosaminyltransferase-14 (GALNT14) is a member of acetylgalactosaminyltransferases family. We have shown that GALNT14 could promote breast cancer cell invasion. However, the underlying molecular mechanism is unclear. Here, using yeast two hybrid, we find that EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) interacts with GALNT14. Both in vitro and in vivo binding assays show that EFEMP2 is associated with GALNT14. Moreover, we find that GALNT14 mediates glycosylation of EFEMP2. EFEMP2 significantly increased the invasion ability of breast cancer cells including MCF-7 and MBA-MD-231 cells, and this phenomenon is suppressed by knockdown expression of GALNT14. In addition, the GALNT14-dependent O-glycosylation of EFEMP-2 regulates the stability of EFEMP-2 protein in breast cancer cells. Taken together, our results demonstrate a novel molecular mechanism underlying breast cancer invasion.     

Down-Regulation of miR-126 Is Associated with Colorectal Cancer Cells Proliferation,Migration and Invasion by Targeting IRS-1 via the AKT and ERK1/2 Signaling Pathways

Background

Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-126 has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126 on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate 1 (IRS-1) and its possible signaling pathway by miR-126.

Methods

The effect of miR-126 on IRS-1, AKT, and ERK1/2 expression was assessed in the CRC cell lines HT-29 and HCT-116 with a miR-126 mimic or inhibitor to increase or decrease miR-126 expression. Furthermore, the roles of miR-126 in regulation of the biological properties of CRC cells were analyzed with miR-126 mimic or inhibitor-transfected cells. The 3′-untranslated region (3′-UTR) of IRS-1 regulated by miR-126 was analyzed by using a dual-luciferase reporter assay.

Results

We found that IRS-1 is the functional downstream target of miR-126 by directly targeting the 3′-UTR of IRS-1. Endogenous miR-126 and exogenous miR-126 mimic inhibited IRS-1 expression. Furthermore, gain-of-function or loss-of-function studies showed that over-expression of miR-126 down-regulated IRS-1, suppressed AKT and ERK1/2 activation, CRC cells proliferation, migration, invasion, and caused cell cycle arrest, but had no effect on cell apoptosis. Knockdown of miR-126 promoted these processes in HCT-116 cells and promoted AKT and ERK1/2 activation by up-regulating the expression of the IRS-1 protein.

Conclusions

MiR-126 may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1 via AKT and ERK1/2 signaling pathways.     

1-硝基-2-酰基蒽醌-缬氨酸通过抑制ERK1/2激活和ERCC1表达诱导结肠癌细胞死亡

化学方法合成是新药研发的一种重要途径。结合抗肿瘤药物的作用机制以及蒽醌类衍生物的构效关系,设计合成了一类新的蒽醌类衍生物1-硝基-2-酰基蒽醌-缬氨酸(简称C3),发现其具有很好的抗肿瘤活性。为了确定蒽醌类衍生物C3对结肠癌HCT116和HT29细胞的作用及其分子机制,首先通过MTT比色法检测C3对结肠癌HCT116和HT29细胞活性的影响。结果显示,C3对这两种结肠癌细胞具有明显的抑制作用,呈时间和剂量依赖性。60μg/mL的C3处理HCT116和HT29细胞48 h,细胞活性分别是50.67%和59.77%,达到了半抑制浓度;同时,其细胞形态和细胞核发生明显变化。进一步采用Western印迹和qRT-PCR技术,检测C3对DNA切除修复交叉互补1(excision repair cross-complementation group 1,ERCC1)转录水平和蛋白质水平表达及其稳定性的影响。结果表明,C3降低了ERCC1转录水平和蛋白质水平的表达,并且减弱了ERCC1转录水平和蛋白质水平的稳定性。最后,用U0126(MEK1/2抑制剂)和C3联合作用结肠癌HCT116和HT29细胞,通过Western印迹检测ERCC1蛋白质水平的表达。结果表明,C3通过降低p-ERK1/2的蛋白质水平的表达,从而抑制ERCC1的表达。上述结果证明,C3通过细胞外调节蛋白激酶(extracellular regulated protein kinases, ERK1/2)信号通路,降低了ERCC1转录水平和蛋白质水平的稳定性,使ERCC1转录水平和蛋白质水平表达发生下调,进而抑制结肠癌HCT116和HT29细胞的活性。     

MiR-519d-3p Suppresses Invasion and Migration of Trophoblast Cells via Targeting MMP-2

Our study was approved by the Medical Ethics Committee of Tang Du Hospital, Fourth Military Medical University and complied strictly with national ethical guidelines. Preeclampsia (PE) is a specific clinical disorder characterized by gestational hypertension and proteinuria and is a leading cause of maternal and perinatal mortality worldwide. The miR-519d-3p is upregulated in the maternal plasma of patients with PE which indicates a possible association between this microRNA and the pathogenesis of PE. No studies to date have addressed the effect of miR-519d-3p on the invasion and migration of trophoblast cells. In our study, we found that miR-519d-3p expression was elevated in placental samples from patients with PE. In vitro, overexpression of miR-519d-3p significantly inhibited trophoblast cell migration and invasion, whereas transfection of a miR-519d-3p inhibitor enhanced trophoblast cell migration and invasion. Luciferase assays confirmed that matrix metalloproteinase-2 (MMP-2) is a direct target of miR-519d-3p. Quantitative real-time PCR and western blot assays showed that overexpression of miR-519d-3p downregulated MMP-2 mRNA and protein expression. Knockdown of MMP-2 using a siRNA attenuated the increased trophoblast migration and invasion promoted by the miR-519d-3p inhibitor. In placentas from patients with PE or normal pregnancies, a negative correlation between the expression of MMP-2 and miR-519d-3p was observed using the Pearson correlation and linear regression analysis. Our present findings suggest that upregulation of miR-519d-3p may contribute to the development of PE by inhibiting trophoblast cell migration and invasion via targeting MMP-2; miR-519d-3p may represent a potential predictive and therapeutic target for PE.