Cell Death of MCF-7 Human Breast Cancer Cells Induced by EGFR Activation in the Absence of Other Growth Factors

The Epidermal Growth Factor (EGF) Receptor (EGFR) plays an important role in the growth and progression of breast cancer. Overexpression of EGFR or the high activity of EGFR signal pathway has been related with increases in cell proliferation and a poor prognosis in patients with breast cancer. Several human breast cancer cell lines depend on estrogen for their proliferation. EGF may bypass the requirement of estrogen for the proliferation of breast cancer cells. To evaluate this hypothesis, MCF-7 breast cancer cells were stimulated with EGF and the effects on cell proliferation, signal pathways, and cell cycle progression were determined. The results demonstrate that EGF stimulation in the absence of others growth factors induced a modest effect on cell proliferation and the induction of a cellular arrest in the G1 phase of the cell cycle. Although phosphorylation of AKT and ERK proteins were detected, this phosphorylation was insufficient to support of cell cycle progression. Cellular arrest in G1 phase was accompanied by an increase in p21CIP1 protein, down regulation of the BCL-2 protein, induction of caspase-8, and ARHI/NOEY2 an imprinted tumor suppressor gene. These results indicate that EGFR activation by itself is not sufficient for the proliferation of breast cancer cells and suggest the existence of a mechanism that induces apoptosis upon EGFR activation.     

EGF Induces Cell Motility and Multi-Drug Resistance Gene Expression in Breast Cancer Cells

The epidermal growth factor receptor (EGFR) is important for normal development, differentiation, and cell proliferation. Deregulation of EGFR has been observed in breast cancer. EGFR and signal pathways activated by these receptors have been associated with an advanced tumor stage and a poor clinical prognosis in breast cancer, however, the precise mechanisms responsible for this process are still not known. Here we show that treatment of MCF-7 breast cancer cells with EGF activated Akt and ERK, induced morphological changes, and increased cell motility. In addition, the constitutive expression of Raf-1 and the use of a MEK inhibitor demonstrated the participation of the Raf/MEK/ERK pathway in these processes. Importantly we detected that EGF induced MRP-1, 3, 5 and 7 gene expression and an increase in MRP1 promoter activity. In conclusion, treatment of MCF-7 breast cancer cells with EGF, in the absence of other growth factors, resulted in activation of EGFR signal transduction pathways; which were related with cell motility and drug resistance.     

干扰TGFβRⅠ增加乳腺癌细胞MDA-MB-231对化疗药物的敏感性

转化生长因子β(transforming growth factorβ,TGFβ)与其受体(transforming growth factorβreceptor,TGFβR)结合激活下游信号通路,在肿瘤的发生发展和转移中具有重要意义,且已有迹象表明该通路可能介导肿瘤的化疗耐受.本研究构建了干扰转化生长因子βⅠ型受体(transforming growth factor receptor typeⅠ,TGFβRⅠ)的重组质粒pRNAT-U6.1/TGFβRⅠ-sh1,发现其可在mRNA和蛋白质水平均显著下调TGFβRⅠ的表达,P0.01.后将该干扰质粒转染乳腺癌细胞MCF-7/5Fu、MCF-7、MDA-MB-231、MDA-MB-453,建立了稳定的乳腺癌TGFβRⅠ干扰模型;MTS法检测上述4种细胞模型对5-氟尿嘧啶(5-fluorouracil,5-FU)和紫杉醇(taxol,TAX)的敏感性.结果显示,MCF-7、MCF-7/5Fu和MDA-MB-453细胞在干扰TGFβRⅠ之后对5-FU和TAX敏感性并未发生改变;但是间质表型的MDA-MB-231细胞在干扰TGFβRⅠ之后对5-FU和TAX的敏感性显著增加.由此可见,干扰TGFβRⅠ的表达阻断TGFβ信号通路,进而显著增加间质型乳腺癌细胞对化疗药物的敏感性,这为临床乳腺癌治疗提供了一定的理论依据.     

Elevated levels of ERK2 in human breast carcinoma MCF-7 cells transfected with protein kinase Cα

Abstract. We investigated the effect of elevated levels of protein kinase Cα (PKCα) on cell proliferation in human breast carcinoma cells (MCF-7). MCF-7 cells transfected with either the pSV₂M(2)6 vector without the insert (MCF-7/Vector) or containing a full length cDNA encoding PKCα (MCF-7/PKCα) were compared. MCF-7/PKCα cells were found to have an increased proliferative rate with a doubling time of 15 h as compared to 42 h for MCF-7/Vector cells. Flow cytometry illustrated a greater percentage of MCF-7/PKCα cells in the S phase of the cell cycle. Western and Northern blot analyses demonstrated an increase in extracellular regulated protein kinase 2 (ERK2) gene expression in MCF-7/PKCα cells but no alteration of this gene expression in MCF-7/Vector cells. These results suggested that the elevated level of ERK2 which is also known as mitogen activated protein kinase is probably involved in the increase in MCF-7/PKCα cell proliferation.     

Signaling pathways regulating aromatase and cyclooxygenases in normal and malignant breast cells

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C(19) androgens to C(18) estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE(2) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression in breast cancer specimens. Knowledge of the signaling pathways that regulate the expression and enzyme activity of aromatase and cyclooxygenases (COXs) in stromal and epithelial breast cells will aid in understanding the interrelationships of these two enzyme systems and potentially identify novel targets for regulation. The effects of epidermal growth factor (EGF), transforming growth factor-beta (TGFbeta), and tetradecanoyl phorbol acetate (TPA) on aromatase and COXs were studied in primary cultures of normal human adipose stromal cells and in cell cultures of normal immortalized human breast epithelial cells MCF-10F, estrogen-responsive human breast cancer cells MCF-7, and estrogen-unresponsive human breast cancer cells MDA-MB-231. Levels of the constitutive COX isozyme, COX-1, were not altered by the various treatments in the cell systems studied. In breast adenocarcinoma cells, EGF and TGFbeta did not alter COX-2 levels at 24h, while TPA induced COX-2 levels by 75% in MDA-MB-231 cells. EGF and TPA in MCF-7 cells significantly increased aromatase activity while TGFbeta did not. In contrast to MCF-7 cells, TGFbeta and TPA significantly increased activity in MDA-MB-231 cells, while only a modest increase with EGF was observed. Untreated normal adipose stromal cells exhibited high basal levels of COX-1 but low to undetectable levels of COX-2. A dramatic induction of COX-2 was observed in the adipose stromal cells by EGF, TGFbeta, and TPA. Aromatase enzyme activity in normal adipose stromal cells was significantly increased by EGF, TGFbeta and TPA after 24h of treatment. In summary, the results of this investigation on the effects of several paracrine and/or autocrine signaling pathways in the regulation of expression of aromatase, COX-1, and COX-2 in breast cells has identified more complex relationships. Overall, elevated levels of these factors in the breast cancer tissue microenvironment can result in increased aromatase activity (and subsequent increased estrogen biosynthesis) via autocrine mechanisms in breast epithelial cells and via paracrine mechanisms in breast stromal cells. Furthermore, increased secretion of prostaglandins such as PGE(2) from constitutive COX-1 and inducible COX-2 isozymes present in epithelial and stromal cell compartments will result in both autocrine and paracrine actions to increase aromatase expression in the tissues.     

SHC-alpha5beta1 integrin interactions regulate breast cancer cell adhesion and motility.

The oncogenic SHC proteins are signaling substrates for most receptor and cytoplasmic tyrosine kinases (TKs) and have been implicated in cellular growth, transformation, and differentiation. In tumor cells overexpressing TKs, the levels of tyrosine phosphorylated SHC are chronically elevated. The significance of amplified SHC signaling in breast tumorigenesis and metastasis remains unknown. Here we demonstrate that seven- to ninefold overexpression of SHC significantly altered interactions of cells with fibronectin (FN). Specifically, in human breast cancer cells overexpressing SHC (MCF-7/SHC) the association of SHC with alpha5beta1 integrin (FN receptor) was increased, spreading on FN was accelerated, and basal growth on FN was reduced. These effects coincided with an early decline of adhesion-dependent MAP kinase activity. Basal motility of MCF-7/SHC cells on FN was inhibited relative to that in several cell lines with normal SHC levels. However, when EGF or IGF-I was used as the chemoattractant, the locomotion of MCF-7/SHC cells was greatly (approx fivefold) stimulated, while it was only minimally altered in the control cells. These data suggest that SHC is a mediator of the dynamic regulation of cell adhesion and motility on FN in breast cancer cells.     

小干扰RNA靶向VEGF基因体内外抑制乳腺癌细胞MCF-7的增殖

 血管生成与肿瘤生长、侵袭、转移密切相关.血管内皮生长因子能特异地促进内皮细胞分裂、增殖及迁移,在肿瘤新生血管生成过程中起着至关重要的作用.通过RNAi抑制VEGF表达的抗血管生成疗法可有效应用于肿瘤治疗.本研究采用化学修饰的siRNA在体内外抑制VEGF基因表达,探讨化学修饰的siRNA介导的RNA干扰技术在乳腺癌基因治疗的可行性和特异性.选用阳离子脂质体Lipofectamine^TM2000作为转染试剂,将针对人VEGF基因的小干扰RNA(small interfering RNA,siRNA)转染人类乳腺细胞株MCF-7和裸鼠移植瘤,在体内外诱导RNAi.采用四甲基偶氮唑蓝（MTT）法,逆转录聚合酶链反应(RT-PCR),蛋白印迹实验等检测siRNA治疗组和对照组VEGF基因表达及细胞增殖变化.体外实验结果显示：靶向VEGF基因siRNA转染乳腺癌MCF-7细胞后,细胞生长抑制率达52.5%;VEGF的mRNA和蛋白表达水平显著降低（P<0.01）;裸鼠体内实验结果显示：siRNA治疗组瘤组织的增长受到明显抑制;RT-PCR结果同时表明治疗组VEGF表达下调.体内外对照组各指标无显著变化.化学修饰的siRNA介导的RNAi在体内外均能成功下调靶基因VEGF的表达,抑制MCF-7细胞增殖,是潜在的肿瘤治疗新方法.     

HIV-1 Tat peptide immunoconjugates differentially sensitize breast cancer cells to selected antiproliferative agents that induce the cyclin-dependent kinase inhibitor p21WAF-1/CIP-1

In this study, we evaluated the ability of anti-p21 antibodies conjugated to 17-mer peptides [GRKKRRQRRRPPQGYGC] harboring the membrane-translocating and nuclear import sequences [underlined] of HIV-1 tat protein to inhibit the cyclin-dependent kinase inhibitor, p21(WAF-1/Cip-1) (p21) and differentially sensitize MDA-MB-468 and MCF-7 human breast cancer (BC) cells to the antiproliferative effects of treatments that induce or do not induce p21. BC cells were treated with increasing concentrations of epidermal growth factor (EGF; 0.5-10 nM), the topoisomerase I inhibitor, camptothecin (CPT; 0.1-4 muM), or increasing doses of gamma-radiation (2-20 Gy). Western blot was used to evaluate p21 expression. The effect of treatment on cell cycle distribution was studied. Growth inhibition was measured by the WST-1 assay. Expression of p21 was increased in MDA-MB-468 cells treated with EGF or CPT but not by gamma-irradiation. MCF-7 cells exhibited p21 upregulation following exposure to CPT and gamma-radiation but not EGF. EGF caused cell cycle arrest in G(1) phase for MDA-MB-468 cells. CPT caused G(1)-phase arrest in MDA-MB-468 cells and prolonged S phase in MCF-7 cells. gamma-Radiation caused an increase in cells in G(2)/M phase for MDA-MB-468 and MCF-7. MDA-MB-468 cells were growth-inhibited by EGF, CPT, and gamma-radiation. MCF-7 cells were growth-stimulated by EGF and inhibited by CPT and gamma-radiation. Combining EGF with tat-anti-p21 immunoconjugates (ICs) amplified the growth-inhibitory effect on MDA-MB-468 cells 1.2-fold to 2.3-fold, but had no effect on the growth stimulation of MCF-7 cells by EGF. Tat-anti-p21 ICs sensitized MCF-7 cells 1.4-fold to gamma-radiation but had no effect on the growth of gamma-irradiated MDA-MB-468 cells. Tat-anti-p21 ICs sensitized both MDA-MB-468 and MCF-7 cells 1.7-fold to CPT. We conclude that tat-anti-p21 ICs are promising sensitizers for cytotoxic cancer therapies and that their sensitization is dependent on treatment-related p21 expression. This general approach could potentially be extended to other growth-regulatory molecules that are associated with tumor growth and progression.     

SHC–α5β1 Integrin Interactions Regulate Breast Cancer Cell Adhesion and Motility

The oncogenic SHC proteins are signaling substrates for most receptor and cytoplasmic tyrosine kinases (TKs) and have been implicated in cellular growth, transformation, and differentiation. In tumor cells overexpressing TKs, the levels of tyrosine phosphorylated SHC are chronically elevated. The significance of amplified SHC signaling in breast tumorigenesis and metastasis remains unknown. Here we demonstrate that seven- to ninefold overexpression of SHC significantly altered interactions of cells with fibronectin (FN). Specifically, in human breast cancer cells overexpressing SHC (MCF-7/SHC) the association of SHC with α5β1 integrin (FN receptor) was increased, spreading on FN was accelerated, and basal growth on FN was reduced. These effects coincided with an early decline of adhesion-dependent MAP kinase activity. Basal motility of MCF-7/SHC cells on FN was inhibited relative to that in several cell lines with normal SHC levels. However, when EGF or IGF-I was used as the chemoattractant, the locomotion of MCF-7/SHC cells was greatly (approx fivefold) stimulated, while it was only minimally altered in the control cells. These data suggest that SHC is a mediator of the dynamic regulation of cell adhesion and motility on FN in breast cancer cells.     

Expression profiling after induction of demethylation in MCF-7 breast cancer cells identifies involvement of TNF-α mediated cancer pathways

Epigenetic methylation change is a major process that occurs during cancer development. Even though many tumor-related genes have been identified based on their relationship between methylation and expression, few studies have been conducted to investigate the relevant biological pathways involved in these changes. To identify essential pathways likely to be affected by methylation in breast cancer, we examined a pool of genes in which expression was upregulated after induction of demethylation by 5-Aza-2′-deoxycytidine (Aza) in the MCF-7 breast cancer cell line. Genome-wide demethylation was confirmed by monitoring the demethylation of a previously known gene, SULT1A1. Overall, 210 and 213 genes were found to be upregulated and downregulated (fold change ≥ 2), respectively, in common in cells treated with 5 and 10 μM of Aza. Network analysis of these 423 genes with altered expression patterns identified the involvement of a cancer related network of genes that were heavily regulated by TNF-α in breast tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to TNF-α-dependent apoptosis may be intimately involved in tumorigenesis in MCF-7 cells.     

Reduced growth rate accompanied by aberrant epidermal growth factor signaling in drug resistant human breast cancer cells

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.     

NDF induces expression of a novel 46 kD protein in estrogen receptor positive breast cancer cells

Most human breast tumors start as estrogen-dependent, but during the course of the disease become refractory to hormone therapy. The transition of breast tumors from estrogen dependent to independent behavior may be regulated by autocrine and/or paracrine growth factor(s) that are independent of the estrogen receptor (ER). We have investigated the role(s) of NDF (neu-differentiation factor) in the biology of estrogen positive breast cancer cells by using MCF-7 cells as a model system. Treatment of MCF-7 cells with human recombinant NDF-β2 (NDF) inhibited the ER expression by 70% and this was associated with growth stimulation in an estrogen-independent manner. To explore the mechanism(s) of action of NDF in MCF-7 cells, we examined the expression of NDF-inducible gene products. We report here that NDF stimulated the levels of expression of a 46 kD protein (p46) (in addition to few minor proteins) in ER positive breast cancer cells including MCF-7, T-47-D, and ZR-75-R cells but not in ER negative breast cancer cells including MDA-231, SK-BP-3, and MDA-468 cells. This effect of NDF was due to induction in the rate of synthesis of new p46. The observed NDF-mediated induction of p46 expression was specific as there was no such effect by epidermal growth factor or 17-β-estradiol, and inclusion of actinomycin D partially inhibited the p46 induction elicited by NDF. NDF-inducible stimulation of p46 expression was an early event (2–6 h) which preceded the period of down-regulation of ER expression by NDF. These results support the existence of NDF-responsive specific cellular pathway(s) that may regulate ER, and these interactions could play a role(s) in hormone-independence of ER positive breast cancer cells. © 1996 Wiley-Liss, Inc.