Mass spectrometry (LC-MS/MS) site-mapping of N-glycosylated membrane proteins for breast cancer biomarkers.
ABSTRACT: Cancer cell membrane proteins are released into the plasma/serum by exterior protein cleavage, membrane sloughing, cellular secretion or cell lysis, and represent promising candidates for interrogation. Because many known disease biomarkers are both glycoproteins and membrane bound, we chose the hydrazide method to specifically target, enrich, and identify glycosylated proteins from breast cancer cell membrane fractions using the LTQ Orbitrap mass spectrometer. Our initial goal was to select membrane proteins from breast cancer cell lines and then to use the hydrazide method to identify the N-linked proteome as a prelude to evaluation of plasma/serum proteins from cancer patients. A combination of steps facilitated identification of the glycopeptides and also defined the glycosylation sites. In MCF-7, MDA-MB-453 and MDA-MB-468 cell membrane fractions, use of the hydrazide method facilitated an initial enrichment and site mapping of 27 N-linked glycosylation sites in 25 different proteins. However, only three N-linked glycosylated proteins, galectin-3 binding protein, lysosome associated membrane glycoprotein 1, and oxygen regulated protein, were identified in all three breast cancer cell lines. In addition, MCF-7 cells shared an additional 3 proteins with MDA-MB-453. Interestingly, the hydrazide method isolated a number of other N-linked glycoproteins also known to be involved in breast cancer, including epidermal growth factor receptor (EGFR), CD44, and the breast cancer 1, and early onset isoform 1 (BRCA1) biomarker. Analyzing the N-glycoproteins from membranes of breast cancer cell lines highlights the usefulness of the procedure for generating a practical set of potential biomarkers.
Project description:The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR) is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1) depolarization responses induced by paxilline (PAX), a specific KCa1.1 blocker and (2) PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory ? and ? subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in breast cancer cells.
Project description:BACKGROUND: Previous research has indicated that at various organ sites there is a subset of adenocarcinomas that is regulated by beta-adrenergic and arachidonic acid-mediated signal transduction pathways. We wished to determine if this regulation exists in breast adenocarcinomas. Expression of mRNA that encodes a G-protein coupled inwardly rectifying potassium channel (GIRK1) has been shown in tissue samples from approximately 40% of primary human breast cancers. Previously, GIRK channels have been associated with beta-adrenergic signaling. METHODS: Breast cancer cell lines were screened for GIRK channels by RT-PCR. Cell cultures of breast cancer cells were treated with beta-adrenergic agonists and antagonists, and changes in gene expression were determined by both relative competitive and real time PCR. Potassium flux was determined by flow cytometry and cell signaling was determined by western blotting. RESULTS: Breast cancer cell lines MCF-7, MDA-MB-361 MDA-MB 453, and ZR-75-1 expressed mRNA for the GIRK1 channel, while MDA-MB-468 and MDA-MB-435S did not. GIRK4 was expressed in all six breast cancer cell lines, and GIRK2 was expressed in all but ZR-75-1 and MDA-MB-435. Exposure of MDA-MB-453 cells for 6 days to the beta-blocker propranolol (1 microM) increased the GIRK1 mRNA levels and decreased beta2-adrenergic mRNA levels, while treatment for 30 minutes daily for 7 days had no effect. Exposure to a beta-adrenergic agonist and antagonist for 24 hours had no effect on gene expression. The beta adrenergic agonist, formoterol hemifumarate, led to increases in K+ flux into MDA-MB-453 cells, and this increase was inhibited by the GIRK channel inhibitor clozapine. The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a high affinity agonist for beta-adrenergic receptors stimulated activation of Erk 1/2 in MDA-MB-453 cells. CONCLUSIONS: Our data suggests beta-adrenergic receptors and GIRK channels may play a role in breast cancer.
Project description:The ADAM proteases are best known for their role in shedding the extracellular domain of transmembrane proteins. Among the transmembrane proteins shed by ADAM10 are notch, HER2, E-cadherin, CD44, L1 and the EGFR ligands, EGF and betacellulin. As cleavage of several of these proteins has been implicated in cancer formation and progression, we hypothesised that ADAM10 is also involved in these processes.ADAM10 expression was decreased by RNA interference and the effects of this on cell numbers, invasion and migration were determined. We also examined the effect of ADAM10 inhibition on breast cancer cell line invasion and migration.Using the triple-negative (TN) breast cancer cell lines, BT20, MDA-MB-231 and the non-TN cell line MDA-MB-453, knockdown of ADAM10 expression significantly decreased in vitro migration (P<0.01; for each cell line). Similarly, treatment with the ADAM10-selective inhibitor GI254023X reduced migration in the three cell lines (for BT20, P<0.001; for MDA-MB-231, P=0.005; for MDA-MB-453, P=0.023). In contrast, neither knockdown of ADAM10 nor treatment with the ADAM10-selective inhibitor GI254023X significantly affected cell numbers. Using extracts of primary breast cancers, higher levels of ADAM10 were found more frequently in high-grade vs low-grade tumours (P<0.001) and in oestrogen receptor (ER)-negative compared with ER-positive tumours (P=0.005). Analysis of pooled publicly available data sets found that high levels of ADAM10 mRNA were associated with adverse outcome in patients with the basal subtype of breast cancer.Based on our combined cell line and breast cancer extract data, we conclude that ADAM10 is likely to be involved in breast cancer progression, especially in the basal subtype.
Project description:Metabolism is an important differentiating feature of cancer cells. Lactate dehydrogenases (LDH) A/B are metabolically important proteins and are involved in the critical step of inter-conversion of lactate to pyruvate. Panepoxydone (PP), a natural NF-kB inhibitor, significantly reduces the oxygen consumption and lactate production of MCF-7 and triple negative (MDA-MB-231, MDA-MB-468 and MDA-MB-453) breast cancer cells. We further observed that PP inhibited mitochondrial membrane potential and the ATP synthesis using flow cytometry. PP also up-regulated LDH-B and down-regulated LDH-A expression levels in all breast cancer cells to similar levels observed in HMEC cells. Over-expression of LDH-B in cancer cell lines leads to enhanced apoptosis, mitochondrial damage, and reduced cell migration. Analyzing the patient data set GDS4069 available on the GEO website, we observed 100% of non TNBC and 60% of TNBC patients had less LDH-B expression than LDH-A expression levels. Herein we report a new term called Glycolytic index, a novel method to calculate utilization of oxidative phosphorylation in breast cancer cells through measuring the ratio of the LDH-B to LDH-A. Furthermore, inhibitors of NF-kB could serve as a therapeutic agent for targeting metabolism and for the treatment of triple negative breast cancer.
Project description:Trastuzumab plus docetaxel is a mainstay to treat HER2-positive breast cancers. However, developing nanoparticles could help to improve the efficacy/toxicity balance of this doublet by improving drug trafficking and delivery to tumors. This project aimed to develop an immunoliposome in breast cancer, combining docetaxel encapsulated in a stealth liposome engrafted with trastuzumab, and comparing its performances on human breast cancer cell lines with standard combination of docetaxel plus trastuzumab.Several strategies to engraft trastuzumab to pegylated liposomes were tested. Immunoliposomes made of natural (antibody nanoconjugate-1 [ANC-1]) and synthetic lipids (ANC-2) were synthesized using standard thin film method and compared in size, morphology, docetaxel encapsulation, trastuzumab engraftment rates and stability. Antiproliferative activity was tested on human breast cancer models ranging from almost negative (MDA-MB-231), positive (MDA-MB-453) to overexpressing (SKBR3) HER2. Finally, cell uptake of ANC-1 was studied by electronic microscopy.ANC-1 showed a greater docetaxel encapsulation rate (73%±6% vs 53%±4%) and longer stability (up to 1 week) as compared with ANC-2. Both ANC presented particle size ?150 nm and showed similar or higher in vitro antiproliferative activities than standard treatment, ANC-1 performing better than ANC-2. The IC50s for docetaxel combined to free trastuzumab were 8.7±4, 2±0.7 and 6±2 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. The IC50s for ANC-1 were 2.5±1, 1.8±0.6 and 3.4±0.8 nM and for ANC-2 were 1.8±0.3 nM, 2.8±0.8 nM and 6.8±1.8 nM with MDA-MB-231, MDA-MB-453 and SKBR3, respectively. Cellular uptake appeared to depend on HER2 expression, the higher the expression, the higher the uptake.In vitro results suggest that higher antiproliferative efficacy and efficient drug delivery can be achieved in breast cancer models using nanoparticles.
Project description:The goal of this study was to determine the transcriptional changes associated with breast cancer cells undergoing vascular mimicry in a 3D assay. Two breast cancer cell lines were plated on matrigel in the presence or absence of serum. MDA-MB-231 cells undergo vascular mimicry on matrigel in the absence of serum, MDA-MB-453 cells do not. Overall design: Four samples were analyzed. MDA-MB-231 and MDA-MB-453 cells were plated for 24 hours on matrigel in the presence or absence of serum. MDA-MB-231 cells undergo vascular mimicry when plated on matrigel in the absence of serum, while MDA-MB-453 cells do not.
Project description:This SuperSeries is composed of the following subset Series: GSE28305: Effect of 5a-dihydrotestosterone on breast cancer cell line MDA-MB-453 GSE28788: Androgen receptor cistrome in breast cancer cell line MDA-MB-453 with 5a-dihydrotestosterone (DHT) stimulation Refer to individual Series
Project description:Androgen receptor (AR) is expressed in 60-70% of breast cancers independent of estrogen receptor (ER) expression, however its function in breast cancer is largely unknown. Our study identified the high level of AR in ER/HER2+ breast tumors and andorgen and AR greatly stimulated growth of MDA-MB-453 breast cancer cells. To define the genome-wide AR binding sites, we performed AR ChIP-seq using MDA-MB-453 breast cancer cells followig stimulation of DHT. We also identified FOXA1 is a crucial AR cofactor in MDA-MB-453 cells and the FOXA cistrome showed signaficant overlap with AR at both early and late time points of DHT stimulation. Overall design: AR ChIP was performed in MDA-MB-453 breast cancer cells following 5a-dihydrotestosterone (DHT) stimulation for 4h and 16h respectively. FOXA1 ChIP-seq was performed after 4h DHT stimulation in MDA-MB-453 cells.
Project description:INTRODUCTION: Tamoxifen, a selective estrogen receptor (ER) modulator, may affect cancer cell survival through mechanisms other than ER antagonism. In the present study, we tested the efficacy of tamoxifen in a panel of ER-negative breast cancer cell lines and examined the drug mechanism. METHODS: In total, five ER-negative breast cancer cell lines (HCC-1937, MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3) were used for in vitro studies. Cellular apoptosis was examined by flow cytometry and Western blot analysis. Signal transduction pathways in cells were assessed by Western blot analysis. The in vivo efficacy of tamoxifen was tested in xenograft nude mice. RESULTS: Tamoxifen induced significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3 cells, but not in HCC-1937 cells. Tamoxifen-induced apoptosis was associated with inhibition of cancerous inhibitor of protein phosphatase 2A (CIP2A) and phospho-Akt (p-Akt) in a dose-dependent manner. Ectopic expression of either CIP2A or Akt protected MDA-MB-231 cells from tamoxifen-induced apoptosis. In addition, tamoxifen increased protein phosphatase 2A (PP2A) activity, and tamoxifen-induced apoptosis was attenuated by the PP2A antagonist okadaic acid in the sensitive cell lines, but not in resistant HCC-1937 cells. Moreover, silencing CIP2A by small interfering RNA sensitized HCC-1937 cells to tamoxifen-induced apoptosis. Furthermore, tamoxifen regulated CIP2A protein expression by downregulating CIP2A mRNA. Importantly, tamoxifen inhibited the in vivo growth of MDA-MB-468 xenograft tumors in association with CIP2A downregulation, whereas tamoxifen had no significant effect on CIP2A expression and anti-tumor growth in HCC-1937 tumors. CONCLUSIONS: Inhibition of CIP2A determines the effects of tamoxifen-induced apoptosis in ER-negative breast cancer cells. Our data suggest a novel "off-target" mechanism of tamoxifen and suggest that CIP2A/PP2A/p-Akt signaling may be a feasible anti-cancer pathway.
Project description:Androgen receptor (AR) is expressed in 60-70% of breast cancers independent of estrogen receptor (ER) expression, however its function in breast cancer is largely unknown. Our study identified the high level of AR in ER–/HER2+ breast tumors and andorgen and AR greatly stimulated growth of MDA-MB-453 breast cancer cells. To define the genome-wide AR binding sites, we performed AR ChIP-seq using MDA-MB-453 breast cancer cells followig stimulation of DHT. We also identified FOXA1 is a crucial AR cofactor in MDA-MB-453 cells and the FOXA cistrome showed signaficant overlap with AR at both early and late time points of DHT stimulation. AR ChIP was performed in MDA-MB-453 breast cancer cells following 5a-dihydrotestosterone (DHT) stimulation for 4h and 16h respectively. FOXA1 ChIP-seq was performed after 4h DHT stimulation in MDA-MB-453 cells.