Transfected MCF-7 Human Breast Cancer Research Articles

Vacuolar H+ ATPase expression and activity is required for Rab27B‐dependent invasive growth and metastasis of breast cancer

The secretory Rab27B small GTPase promotes invasive growth and metastasis in estrogen receptor (ER) α-positive breast cancer cells by orchestrating the peripheral targeting of vesicles secreting proinvasive growth regulators. Increased Rab27B expression is associated with poor prognosis in breast cancer patients. The molecular mechanisms of peripheral Rab27B secretory vesicle distribution are poorly understood. Mass spectrometry analysis on green fluorescent protein (GFP)-Rab27B vesicles prepared from GFP-Rab27B transfected MCF-7 human breast cancer cells detected eight subunits of the vacuolar H(+)-ATPase (V-ATPase) and the presence of V0a1 and V0d1 subunits was confirmed by Western blot analysis. Reversible inhibition of V-ATPase activity by bafilomycin A1 or transient silencing of V0a1 or V0d1 subunits demonstrated that V-ATPase controls peripheral localization and size of Rab27B vesicles. V-ATPase expression and activity further controls Rab27B-induced collagen type I invasion, cell-cycle progression and invasive growth in the chorioallantoic membrane assay. In agreement, Rab27B-dependent extracellular heat shock protein90α release and matrix metalloprotease-2 activation is markedly reduced by bafilomycin A1 and transient silencing of V0a1 and V0d1 subunits. Poor prognosis ERα-positive primary breast tumors expressing high levels of Rab27B also expressed multiple V-ATPase subunits and showed a strong cytoplasmic and peripheral V-ATPase V1E expression. In conclusion, inhibiting V-ATPase activity by interfering agents and drugs might be an effective strategy for blocking Rab27B-dependent proinvasive secretory vesicle trafficking in ERα-positive breast cancer patients.

Synergistic Inhibition with a Dual Epidermal Growth Factor Receptor/HER-2/neu Tyrosine Kinase Inhibitor and a Disintegrin and Metalloprotease Inhibitor

The ErbB family of receptors is overexpressed in numerous human tumors. Overexpression correlates with poor prognosis and resistance to therapy. Use of ErbB-specific antibodies to the receptors (Herceptin or Erbitux) or ErbB-specific small-molecule inhibitors of the receptor tyrosine kinase activity (Iressa or Tarceva) has shown clinical efficacy in several solid tumors. An alternative method of affecting ErbB-initiated tumor growth and survival is to block sheddase activity. Sheddase activity is responsible for cleavage of multiple ErbB ligands and receptors, a necessary step in availability of the soluble, active form of the ligand and a constitutively activated ligand-independent receptor. This sheddase activity is attributed to the ADAM (a disintegrin and metalloprotease) family of proteins. ADAM 10 is the main sheddase of epidermal growth factor (EGF) and HER-2/neu cleavage, whereas ADAM17 is required for cleavage of additional EGF receptor (EGFR) ligands (transforming growth factor-alpha, amphiregulin, heregulin, heparin binding EGF-like ligand). This study has shown that addition of INCB3619, a potent inhibitor of ADAM10 and ADAM17, reduces in vitro HER-2/neu and amphiregulin shedding, confirming that it interferes with both HER-2/neu and EGFR ligand cleavage. Combining INCB3619 with a lapatinib-like dual inhibitor of EGFR and HER-2/neu kinases resulted in synergistic growth inhibition in MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Combining the INCB7839 second-generation sheddase inhibitor with lapatinib prevented the growth of HER-2/neu-positive BT474-SC1 human breast cancer xenografts in vivo. These results suggest that there may be an additional clinical benefit of combining agents that target the ErbB pathways at multiple points.

Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration

Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D. Ravid, S. Maor, H. Werner, M. Liscovitch, Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling, Oncogene 24 (2005) 1338–1347.]. We now show, using differential detergent extraction, SDS-PAGE and mass spectrometry, that the major protein in the pp340 band is the actin filament cross-linking protein filamin A. The identity of pp340 as filamin A was confirmed by immunoprecipitation of pp340 with specific filamin A antibodies. RT-PCR, flow cytometry and Western blot analyses show that filamin A mRNA and protein levels are respectively 3.5- and 2.5-fold higher in MCF-7/Cav1 cells than in MCF-7 cells. Basal filamin A phosphorylation on Ser-2152, normalized to total filamin A levels, is 7.8-fold higher in MCF-7/Cav1 than in MCF-7 cells. Insulin-like growth factor-I (IGF-I) stimulates phosphorylation of filamin A on Ser-2152 in MCF-7 cells and further enhances Ser-2152 phosphorylation over its already high basal level in MCF-7/Cav1 cells. The effect of IGF-I is inhibited by the PI3K inhibitor wortmannin, indicating that IGF-I-stimulated phosphorylation of filamin A occurs via the PI3K/Akt pathway. Co-immunoprecipitation experiments have confirmed a previous report showing that filamin A and caveolin-1 co-exist in a complex and have revealed the presence of active phospho-Akt in this complex. Ser-2152 phosphorylation of filamin A has been implicated in cancer cell migration. Accordingly, caveolin-1 expression dramatically enhances IGF-I-dependent MCF-7 cell migration. These data indicate that caveolin-1 specifies filamin A as a novel target for Akt-mediated filamin A Ser-2152 phosphorylation thus mediating the effects of caveolin-1 on IGF-I-induced cancer cell migration.

Combining flavopiridol with various signal transduction inhibitors

Treatment of human tumors with a combination of chemotherapeutic agents results in improved response as well as the ability to use less toxic concentrations of the drugs. Recent phase I clinical trials with the cyclin-dependent kinase inhibitor, flavopiridol, have shown some promise in the treatment of a variety of human tumors. Because of the severe toxicity, however, the use of less toxic doses in combination with other antiproliferative agents would be desirable. The purpose of this study was to examine the effects of combining flavopiridol with several signal transduction inhibitors: the SC236 COX-2 inhibitor, a PKC kinase inhibitor and LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor in a control vector transfected MCF-7 human breast cancer cell line (MCF/neo) and a HER-2/neu transfected MCF-7 cell line (MCF/18). Enhanced (better than that seen with either agent alone but not additive) growth inhibition was observed in both cell lines with the combination of flavopiridol and the PKC kinase inhibitor. The combination of flavopiridol and the SC236 COX-2 inhibitor resulted in an enhanced effect in the MCF/18 cell line and a synergistic effect in the MCF/neo cells. The combination of flavopiridol and LY294002 resulted in a synergistic effect in the MCF/18 cell line and an additive effect in the MCF/neo cells. These data suggest that combinations of flavopiridol and signal transduction inhibitors warrant further studies as treatments for breast tumors, and that HER-2/neu expression may influence the choice of inhibitor to combine with flavopiridol.

Increased thioredoxin-1 inhibits SSAT expression in MCF-7 human breast cancer cells

Spermidine/spermine N 1-acetyltransferase (SSAT) regulates polyamine catabolism. Thioredoxin-1 (Trx-1) is a redox protein that is overexpressed in human cancer leading to increased cell proliferation, decreased apoptosis, and decreased patient survival. We report that SSAT mRNA expression is decreased in Trx-1 transfected MCF-7 human breast cancer cells. There is also a decrease in SSAT enzyme activity and lower putrescine levels but no change in spermine or spermidine levels. The expression of SSAT is regulated by the NF-E2-related factor 2 (Nrf-2) and polyamine modulated factor-1 (PMF-1) transcription factor complex. Trx-1 transfected MCF-7 cells showed decreased Nrf-2/PMF-1 DNA binding without a change in Nrf-2 or PMF-1 protein expression. The results suggest that Trx-1 may play a role in the redox regulation of SSAT expression and polyamine homeostasis that could contribute to the biological effects of Trx-1.

Decreased response to paclitaxel versus docetaxel in HER-2/neu transfected human breast cancer cells.

Taxanes are effective in the treatment of metastatic breast cancer. Docetaxel has been shown to be more potent than paclitaxel in inducing bcl-2 phosphorylation and apoptosis and is clinically active in some paclitaxel-resistant breast tumors. HER-2/neu overexpression has been shown to correlate with resistance to hormonal therapy as well as chemotherapy. Using a HER-2/neu transfected MCF-7 human breast cancer cell line, we investigated the role of HER-2/neu overexpression on resistance to paclitaxel and docetaxel treatment. A control vector transfected MCF-7 human breast cancer cell line (MCF/neo) and a HER-2/neu transfected MCF-7 line (MCF/18) were treated with various concentrations of docetaxel or paclitaxel. Cell number was assessed using the MTT tetrazolium dye assay. In the control vector transfected MCF/neo cell line, paclitaxel and docetaxel gave similar dose-dependent growth inhibition ( p = 0.175). In HER-2/neu transfected MCF/18 cells, docetaxel treatment resulted in a dose-dependent inhibition similar to that seen in MCF/neo cells. Paclitaxel, however, gave significantly less growth inhibition than docetaxel in the HER-2/neu overexpressing MCF/18 cells (p = 0.0003). These data suggest that HER-2/neu overexpression may contribute to paclitaxel resistance. In contrast, the cytotoxic effects of docetaxel in these breast carcinoma cells are not affected by HER-2/neu expression. Therefore, docetaxel may be the preferred taxane therapy in HER-2/neu overexpressing breast tumors.

The Aryl Hydrocarbon Receptor Interacts with Estrogen Receptor Alpha and Orphan Receptors COUP-TFI and ERRα1

The molecular mechanisms underlying the apparent “cross-talk” between estrogen receptor (ER)- and arylhydrocarbon receptor (AHR)-mediated activities are unknown. To determine how AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may inhibit ER action and, conversely, to examine how 17-β-estradiol (E2) affects AHR activity, we examined discrete activities of each receptor, i.e., protein–protein interactions, DNA binding, and transcriptional activation. We report that AHR interacts directly with ERα, COUP-TF, and ERRα1, in a ligand-specific manner in vitro. Unoccupied or β-napthoflavone (β-NF)-occupied AHR showed stronger interaction with ERα, COUP-TF, and ERRα1 than when AHR was occupied by the partial antagonist α-naphthoflavone (α-NF), indicating a role for ligand in AHR interaction with these proteins. We also report that AHR interacts with COUP-TF in transfected CV-1 cells. In contrast, the AHR nuclear translocator protein (ARNT) did not interact with COUP-TF, ERRα1, or ERα. We next examined the interaction of either ERα or COUP-TF with a consensus xenobiotic response element (XRE). Purified ERα did not bind the consensus XRE, but COUP-TFI bound the consensus XRE, suggesting a role for COUP-TF as a AHR/ARNT competitor for XRE binding. In transiently transfected MCF-7 human breast cancer cells, overexpression of COUP-TFI inhibited TCDD-activated reporter gene activity from the CYP1A1 promoter. TCDD inhibited estradiol (E2)-activated reporter gene activity from a consensus ERE and from the EREs in the pS2 and Fos genes, and COUP-TFI did not block the antiestrogenic activity of TCDD. The specific interaction of COUP-TF with XREs and AHR together with the inhibition of TCDD-induced gene expression by COUP-TF suggests that COUP-TF may regulate AHR action both by direct DNA binding competition and through protein–protein interactions.

The aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements

To determine the molecular mechanisms underlying the “cross talk” between the activity of 2,3,7,8-tetra-chlorodibenzo- p-dioxin (TCDD), which binds to arylhydrocarbon receptor (AHR) and estradiol (E 2)-liganded estrogen receptor (ER), we first examined the initial step of estrogen action, ligand binding to ER. None of the AHR ligands tested, i.e. TCDD, benzo[a]pyrene, 3,3′,4,4′,5-pentachlorobiphenyl, β-naphthoflavone, or α-naphthoflavone, bound to ERα. We report the first examination of TCDD interaction with ERβ: TCDD did not displace E 2 from ERβ. We then examined a second possible mechanism, i.e. direct inhibition of ERα binding to estrogen response elements (EREs) by the AHR/AHR nuclear translocator (ARNT) complex. The AHR/ARNT heterodimer did not bind either a full or half-site ERE. However, AHR/ARNT bound specifically to oligomers containing naturally occurring EREs derived from the human c- fos, pS2, and progesterone receptor (PR) gene promoters that include xenobiotic response element (XRE)-like sequences. In contrast, neither purified E 2-liganded-ER from calf uterus or recombinant human ERα bound a consensus XRE. TCDD inhibited E 2-activated reporter gene activity from a consensus ERE and from EREs in the pS2, PR, and Fos genes in transiently transfected MCF-7 human breast cancer cells. However, this inhibition was not reciprocal since E 2 did not inhibit TCDD-stimulated luciferase activity from the CYP1A1 promoter in transiently transfected MCF-7 or human endometrial carcinoma HEC-1A cells. We propose that at least part of the mechanism by which the AHR/ARNT complex inhibits estrogen action is by competitively inhibiting ERα binding to imperfect ERE sites, adjacent to or overlapping XREs.

Molecular aspects of estrogen receptor variants in breast cancer.

Measurements of the estrogen receptor (ER) and the estrogen-induced progesterone receptor (PgR) are used by most clinicians as indicators of both overall prognosis and likelihood of response to endocrine therapy. Patients with ER+/PgR+ tumors have the highest likelihood of response; conversely, patients with ER-/PgR-tumors have the lowest likelihood of response. Unfortunately, most patients treated successfully with endocrine therapy eventually develop endocrine-resistant disease recurrence. In an effort to study potential mechanisms of endocrine resistance, we have studied discordant ER-/PgR+ tumors, in which the normally estrogen-regulated PgR gene is induced in the apparent absence of ER. Our laboratory has previously cloned, from ER-/PgR+ tumors, a variant ER mRNA precisely missing the sequence corresponding to ER exon 5, and has demonstrated that the truncated protein product translated from this variant RNA is capable of constitutively inducing the expression of an estrogen-responsive reporter gene in a yeast expression vector system (Fuqua et al. Cancer Res 51:105-109, 1991). In the present report we describe further experiments to characterize the activity and biological consequences of expression of this variant ER in human breast cancer cells. We have stably transfected MCF-7 human breast cancer cells with a mammalian expression vector for the exon 5 deletion variant ER. These transfected cells produce a truncated ER protein of the expected 40 kDa size. Cells expressing the exon 5 ER deletion variant constitutively express PgR, and manifest increased anchorage-independent colony formation in the absence of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)