Prolactin-dependent up-regulation of BRCA1 expression in human breast cancer cell lines.

We report experimental evidence that BRCA1, a breast and ovarian cancer susceptibility gene, is up-regulated in response to prolactin (PRL) stimulation. Expression of the BRCA1 gene was monitored in 2 human breast cancer cell lines (MCF-7 and T-47D) and in the normal mammary epithelial cell line MCF10a. Using competitive RT-PCR, we have shown that PRL induced an increase in BRCA1 mRNA level in MCF-7 and T-47D cell lines at a dose resulting in the maximal enhancement of cell proliferation. The up-regulation was 12-fold in MCF-7 cells and 2-fold in T-47D cells. No increase in BRCA1 mRNA level was observed in the MCF10a cell line. The level of BRCA1 protein was quantified using an affinity chromatography strategy. At the protein level, PRL treatment induced a 4-fold increase of BRCA1 protein expression in MCF-7 and a 6-fold increase in T-47D cells, whereas BRCA1 protein expression was not affected by PRL in MCF10a.     

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 μM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.     

Expression profiling by whole-genome microarray hybridization reveals differential gene expression in breast cancer cell lines after lycopene exposure

The correlation between diet and variation in gene-expression is an important field which could be considered to approach cancer pathways comprehension. We examined the effects of lycopene on breast cancer cell lines using pangenomic arrays. Lycopene is derived predominantly from tomatoes and tomato products and there is some epidemiologic evidence for a preventive role in breast cancer. Previously, we investigated lycopene in breast cancer using a dedicated breast cancer microarray. To confirm these results and explore pathways other than those implicated in breast cancer, for this study we used pangenomic arrays containing 25,000 oligonucleotides. This in vitro study assayed two human mammary cancer cell lines (MCF-7 and MDA-MB-231), and a fibrocystic breast cell line (MCF-10a) treated or not with 10 microM lycopene for 48 h. A competitive hybridization was performed between Cy3-labeled lycopene treated RNA and Cy5-labeled untreated RNA to define differentially expressed genes. Using t-test analysis, a subset of 391 genes was found to be differentially modulated by lycopene between estrogen-positive cells (MCF-7) and estrogen-negative cells (MDA-MB-231, MCF-10a). Hierarchical clustering revealed 726 discriminatory genes between breast cancer cell lines (MCF-7, MDA-MB-231) and the fibrocystic breast cell line (MCF-10a). Modified gene expression was observed in various molecular pathways, such as apoptosis, cell communication, MAPK and cell cycle as well as xenobiotic metabolism, fatty acid biosynthesis and gap junctional intercellular communication.     

Induction of proinflammatory and chemokine genes by lipopolysaccharide and paclitaxel (Taxol) in murine and human breast cancer cell lines

In murine macrophages, the anti-tumor agent, paclitaxel, induces expression of a wide variety of inflammatory and anti-inflammatory genes, and causes cytokine secretion via signaling pathways that overlap with those engaged by lipopolysaccharide (LPS), the endotoxic component of Gram-negative bacteria. Using semi-quantitative RT-PCR for detection of gene expression, coupled with ELISA for the detection of secreted gene products, we analyzed the responsiveness of an extensive panel of cytokine and non-cytokine genes to induction by paclitaxel and LPS in the murine DA-3 breast cancer line. A subset of the genes examined (e.g., G-CSF, MIP-2, iNOS, and IL-1 beta, and GM-CSF) was upregulated >3-20-fold by both LPS and paclitaxel in the DA-3 cell line, while IP-10 mRNA was induced by paclitaxel, but not by LPS. In the human MDA-MB-231 breast cancer cell line, LPS also increased mRNA levels for both GM-CSF and IP-10 significantly, while, paclitaxel increased IP-10 mRNA levels with delayed kinetics and failed to induce GM-CSF mRNA. Co-cultures of murine breast cancer cells and macrophages, stimulated with IFN-gamma plus either paclitaxel or LPS, resulted in augmented release of nitric oxide. As both GM-CSF and IP-10 have been implicated in tumor rejection in vivo through either indirect actions on the host immune system or by inhibiting tumor angiogenesis, our data strengthen the hypothesis that tumor cell-derived inflammatory mediators may, in part, underlie the anti-tumor efficacy of paclitaxel in breast cancer.     

Regulation of progesterone receptor mRNA by oestradiol and antioestrogens in breast cancer cell lines

The induction of progesterone receptor mRNA by oestradiol and antioestrogens has been characterised in the MCF-7 breast cancer cell line. Progesterone receptor mRNA was induced more than 100-fold by oestradiol. The induction was half-maximal in the presence of 10(-10) M oestradiol and maximum levels were reached after 24 h treatment. Progesterone receptor mRNA was induced to 10% of the oestrogen-induced level by tamoxifen and its metabolite 4'-hydroxytamoxifen. The increase was half-maximal in the presence of 5 X 10(-8) M tamoxifen or 5 X 10(-10) M 4'-hydroxytamoxifen. In contrast, neither the benzothiophene antioestrogen LY117018 nor the 7 alpha-alkyl steroidal antioestrogen ICI 164,384 had any effect on progesterone receptor mRNA. The progesterone receptor mRNA was also induced by oestrogen in a T47D subline and in two other oestrogen-responsive breast cancer cell lines (ZR-75, EFM-19). Tamoxifen was a partial oestrogen for progesterone receptor mRNA induction in each of these cell lines. The large induction of the progesterone receptor mRNA by oestrogen in all 4 breast cancer cell lines supports the contention that the progesterone receptor may be a good predictive marker of hormonal response in human breast cancer.     

Expression of progesterone receptor isoforms A and B is differentially regulated by estrogen in different breast cancer cell lines

Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E(2)), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E(2) induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E(2) was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E(2) in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E(2) in a distinct way.     

BRCA1 inhibits membrane estrogen and growth factor receptor signaling to cell proliferation in breast cancer

BRCA1 mutations and estrogen use are risk factors for the development of breast cancer. Recent work has identified estrogen receptors localized at the plasma membrane that signal to cell biology. We examined the impact of BRCA1 on membrane estrogen and growth factor receptor signaling to breast cancer cell proliferation. MCF-7 and ZR-75-1 cells showed a rapid and sustained activation of extracellular signal-related kinase (ERK) in response to estradiol (E2) that was substantially prevented by wild-type (wt) but not mutant BRCA1. The proliferation of MCF-7 cells induced by E2 was significantly inhibited by PD98059, a specific ERK inhibitor, or by dominant negative ERK2 expression and by expression of wt BRCA1 (but not mutant BRCA1). E2 induced the synthesis of cyclins D1 and B1, the activity of cyclin-dependent kinases Cdk4 and CDK1, and G(1)/S and G(2)/M cell cycle progression. The intact tumor suppressor inhibited all of these. wt BRCA1 also inhibited epidermal growth factor and insulin-like growth factor I-induced ERK and cell proliferation. The inhibition of ERK and cell proliferation by BRCA1 was prevented by phosphatase inhibitors and by interfering RNA knockdown of the ERK phosphatase, mitogen-activated kinase phosphatase 1. Our findings support a novel tumor suppressor function of BRCA1 that is relevant to breast cancer and identify a potential interactive risk factor for women with BRCA1 mutations.     

TFF2 (trefoil family factor2) inhibits apoptosis in breast and colorectal cancer cell lines

We have recently demonstrated that human TFF2 inhibits apoptosis in the non-TFF2 expressing breast adenocarcinoma cell line MCF-7. In this study we examined the impact of TFF2 and an anti-TFF2 antibody (hSP3) on the survival of other human adenocarcinoma cell lines; TFF2-positive (LS174T and SW480) and TFF2-negative (MCF-7 and T47D). Addition of TFF2 protected the (TFF2-) lines but had no effect on those constitutively expressing TFF2. Blocking with hSP3 significantly increased apoptosis in the (TFF2+) cell lines with minimal effect on the (TFF2-) cells. Our results show that the cytoprotective effect of TFF2 seen in MCF-7 cells is not cell line-specific and can be abrogated by inhibition of its expression.     

The colony-stimulating factor-1 (CSF-1) receptor sustains ERK1/2 activation and proliferation in breast cancer cell lines

Breast cancer is the second leading cause of cancer-related deaths in western countries. Colony-Stimulating Factor-1 (CSF-1) and its receptor (CSF-1R) regulate macrophage and osteoclast production, trophoblast implantation and mammary gland development. The expression of CSF-1R and/or CSF-1 strongly correlates with poor prognosis in several human epithelial tumors, including breast carcinomas. We demonstrate that CSF-1 and CSF-1R are expressed, although at different levels, in 16/17 breast cancer cell lines tested with no differences among molecular subtypes. The role of CSF-1/CSF-1R in the proliferation of breast cancer cells was then studied in MDAMB468 and SKBR3 cells belonging to different subtypes. CSF-1 administration induced ERK1/2 phosphorylation and enhanced cell proliferation in both cell lines. Furthermore, the inhibition of CSF-1/CSF-1R signaling, by CSF-1R siRNA or imatinib treatment, impaired CSF-1 induced ERK1/2 activation and cell proliferation. We also demonstrate that c-Jun, cyclin D1 and c-Myc, known for their involvement in cell proliferation, are downstream CSF-1R in breast cancer cells. The presence of a proliferative CSF-1/CSF-1R autocrine loop involving ERK1/2 was also found. The wide expression of the CSF-1/CSF-1R pair across breast cancer cell subtypes supports CSF-1/CSF-1R targeting in breast cancer therapy.     

siRNA-mediated knockdown of aryl hydrocarbon receptor nuclear translocator 2 affects hypoxia-inducible factor-1 regulatory signaling and metabolism in human breast cancer cells

Recent human studies found that the mRNA expression level of aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) was positively associated with the prognosis of breast cancer. In this study, we used small interfering RNA techniques to knockdown ARNT2 expression in MCF7 human breast cancer cells, and found that an almost 40% downregulation of ARNT2 mRNA expression increased the expression of sensitive to apoptosis gene (3.36-fold), and decreased the expression of von Hippel-Lindau (0.27-fold) and matrix metalloproteinase-1 (0.35-fold). The metabolite analysis revealed the contents of glucose, glycine, betaine, phosphocholine, pyruvate and lactate involved in the hypoxia-inducible factor (HIF)-1-dependent glycolytic pathway were significantly lower in cells treated with siARNT2. Our results suggested that ARNT2 might play an important role in the modulation of HIF-1-regulated signaling and metabolism.     

Contribution of BRCA1 and BRCA2 mutations to breast and ovarian cancer in Pakistan

The population of Pakistan has been reported to have the highest rate of breast cancer of any Asian population (excluding Jews in Israel) and one of the highest rates of ovarian cancer worldwide. To explore the contribution that genetic factors make to these high rates, we have conducted a case-control study of 341 case subjects with breast cancer, 120 case subjects with ovarian cancer, and 200 female control subjects from two major cities of Pakistan (Karachi and Lahore). The prevalence of BRCA1 or BRCA2 mutations among case subjects with breast cancer was 6.7% (95% confidence interval [CI] 4.1%-9.4%), and that among case subjects with ovarian cancer was 15.8% (95% CI 9.2%-22.4%). Mutations of the BRCA1 gene accounted for 84% of the mutations among case subjects with ovarian cancer and 65% of mutations among case subjects with breast cancer. The majority of detected mutations are unique to Pakistan. Five BRCA1 mutations (2080insA, 3889delAG, 4184del4, 4284delAG, and IVS14-1A-->G) and one BRCA2 mutation (3337C-->T) were found in multiple case subjects and represent candidate founder mutations. The penetrance of deleterious mutations in BRCA1 and BRCA2 is comparable to that of Western populations. The cumulative risk of cancer to age 85 years in female first-degree relatives of BRCA1-mutation-positive case subjects was 48% and was 37% for first-degree relatives of the BRCA2-mutation-positive case subjects. A higher proportion of case subjects with breast cancer than of control subjects were the progeny of first-cousin marriages (odds ratio [OR] 2.1; 95% CI 1.4-3.3; P=.001). The effects of consanguinity were significant for case subjects with early-onset breast cancer (age <40 years) (OR=2.7; 95% CI 1.5-4.9; P=.0008) and case subjects with ovarian cancer (OR=2.4; 95% CI 1.4-4.2; P=.002). These results suggest that recessively inherited genes may contribute to breast and ovarian cancer risk in Pakistan.     

Microarray analyses of hypoxia-regulated genes in an aryl hydrocarbon receptor nuclear translocator (Arnt)-dependent manner

We investigated hypoxia-inducible factor (HIF)-dependent changes in the expression of 5592 genes in response to hypoxia (0.1% O(2), 16 h) by performing cDNA microarray analyses of mouse hepa1c1c7 and BpRc1 cells. BpRc1 cells are a hepa1c1c7 variant defective in HIF-beta/aryl hydrocarbon receptor nuclear translocator (Arnt), and are therefore unable to induce HIF target genes in response to hypoxia. By comparing hepa1c1c7 cells with BpRc1 cells, we were able to investigate hypoxia-regulated gene expression as well as the role played by HIF in regulating the hypoxic-dependent response of gene expression. This study identified 50 hypoxia-induced genes and 36 hypoxia-repressed genes. Quantitative PCR analysis of nine genes confirmed our ability to accurately analyze changes in hypoxia-induced gene expression by microarray analysis. By comparing quantitative PCR analyses of these nine genes in BpRc1 and hepa1c1c7 cells, we determined that eight of the nine hypoxia-induced genes are Arnt dependent. Additional quantitative PCR analyses of eight hypoxia-repressed genes confirmed, with a 50% probability, that microarray analysis was able to predict hypoxia-repressed gene expression. Only two of the four confirmed genes were found to be repressed in an Arnt-dependent manner. Collectively, six of these 13 genes (46.2% probability) showed a pattern of expression consistent with the microarray analysis with regard to Arnt dependence. Finally, we investigated the HIF-1alpha dependence of these 13 genes by quantitative PCR analysis in HIF-1alpha knockdown 3T3-L1 cells. These analyses identified novel hypoxia-regulated genes and confirmed the role of Arnt and HIF-1alpha in regulating their expression. These results identify additional HIF target genes and provide a more complete understanding of hypoxia signaling.     

Expression of plasma membrane calcium pump isoform mRNAs in breast cancer cell lines

The plasma membrane Ca²⁺ ATPase (PMCA) is an important regulator of free intracellular calcium, with dynamic regulation in the rat mammary gland during lactation. Recent studies suggest that Ca²⁺ plays a role in cellular proliferation. To determine if PMCA expression is altered in tumorigenesis, we compared relative levels of PMCA1 mRNA. We found that the relative expression of PMCA1 mRNA is increased, by approximately 270% and 170%, in MCF-7 and MDA-MB-231 human breast cancer cell lines deprived of serum for 72 h, respectively, compared to the similarly treated MCF-10A human mammary gland epithelial cell line. Characterization of PMCA mRNA isoforms revealed that PMCA1b and PMCA4 mRNA are expressed in MCF-7, MDA-MB-231, SK-BR-3, ZR-75-1 and BT-483 breast cancer cell lines. We also detected PMCA2 mRNA expression in all the breast cancer cell lines examined. However, PMCA3 mRNA was only detected in BT-483 cells. Our results suggest that PMCA expression may be altered in breast cancer cell lines, suggesting altered Ca²⁺ regulation in these cell lines. Our results also indicate that breast cancer cell lines can express mRNAs for a variety PMCA isoforms.     

Systematic expression analysis of genes related to multidrug-resistance in isogenic docetaxel- and adriamycin-resistant breast cancer cell lines

Docetaxel (Doc) and adriamycin (Adr) are two of the most effective chemotherapeutic agents in the treatment of breast cancer. However, their efficacy is often limited by the emergence of multidrug resistance (MDR). The purpose of this study was to investigate MDR mechanisms through analyzing systematically the expression changes of genes related to MDR in the induction process of isogenic drug resistant MCF-7 cell lines. Isogenic resistant sublines selected at 100 and 200 nM Doc (MCF-7/100 nM Doc and MCF-7/200 nM Doc) or at 500 and 1,500 nM Adr (MCF-7/500 nM Adr and MCF-7/1,500 nM) were developed from human breast cancer parental cell line MCF-7, by exposing MCF-7 to gradually increasing concentrations of Doc or Adr in vitro. Cell growth curve, flow cytometry and MTT cytotoxicity assay were preformed to evaluate the MDR characteristics developed in the sublines. Some key genes on the pathways related to drug resistance (including drug-transporters: MDR1, MRP1 and BCRP; drug metabolizing-enzymes: CYP3A4 and glutathione S-transferases (GST) pi; target genes: topoisomerase II (TopoIIα) and Tubb3; apoptosis genes: Bcl-2 and Bax) were analyzed at RNA and protein expression levels by real time RT-qPCR and western blot, respectively. Compared to MCF-7/S (30.6 h), cell doubling time of MCF-7/Doc (41.6 h) and MCF-7/Adr (33.8 h) were both prolonged, and the cell proportion of resistant sublines in G1/G2 phase increased while that in S-phase decreased. MCF-7/100 nM Doc and MCF-7/200 nM Doc was 22- and 37-fold resistant to Doc, 18- and 32-fold to Adr, respectively. MCF-7/500 nM Adr and MCF-7/1,500 nM Adr was 61- and 274-fold resistant to Adr, three and 12-fold to Doc, respectively. Meantime, they also showed cross-resistance to the other anticancer drugs in different degrees. Compared to MCF-7/S, RT-qPCR and Western blot results revealed that the expression of MDR1, MRP1, BCRP, Tubb3 and Bcl-2 were elevated in both MCF-7/Doc and MCF-7/Adr, and TopoIIα, Bax were down-regulated in both the sublines, while CYP3A4, GST pi were increased only in MCF-7/Doc and MCF-7/Adr respectively. Furthermore, the changes above were dose-dependent. The established MCF-7/Doc or MCF-7/Adr has the typical MDR characteristics, which can be used as the models for resistance mechanism study. The acquired process of MCF-7/S resistance to Doc or Adr is gradual, and is complicated with the various pathways involved in. There are some common resistant mechanisms as well as own drug-specific changes between both the sublines.     

Distinct genes related to drug response identified in ER positive and ER negative breast cancer cell lines

Breast cancer patients have different responses to chemotherapeutic treatments. Genes associated with drug response can provide insight to understand the mechanisms of drug resistance, identify promising therapeutic opportunities, and facilitate personalized treatment. Estrogen receptor (ER) positive and ER negative breast cancer have distinct clinical behavior and molecular properties. However, to date, few studies have rigorously assessed drug response genes in them. In this study, our goal was to systematically identify genes associated with multidrug response in ER positive and ER negative breast cancer cell lines. We tested 27 human breast cell lines for response to seven chemotherapeutic agents (cyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, and paclitaxel). We integrated publicly available gene expression profiles of these cell lines with their in vitro drug response patterns, then applied meta-analysis to identify genes related to multidrug response in ER positive and ER negative cells separately. One hundred eighty-eight genes were identified as related to multidrug response in ER positive and 32 genes in ER negative breast cell lines. Of these, only three genes (DBI, TOP2A, and PMVK) were common to both cell types. TOP2A was positively associated with drug response, and DBI was negatively associated with drug response. Interestingly, PMVK was positively associated with drug response in ER positive cells and negatively in ER negative cells. Functional analysis showed that while cell cycle affects drug response in both ER positive and negative cells, most biological processes that are involved in drug response are distinct. A number of signaling pathways that are uniquely enriched in ER positive cells have complex cross talk with ER signaling, while in ER negative cells, enriched pathways are related to metabolic functions. Taken together, our analysis indicates that distinct mechanisms are involved in multidrug response in ER positive and ER negative breast cells.