Quantitative Proteomic Profiling Identifies a Potential Novel Chaperone Marker in Resistant Breast Cancer.

Development of aromatase inhibitor resistant breast cancer among postmenopausal women continues to be a major clinical obstacle. Previously, our group demonstrated that as breast cancer cells transition from hormone-dependent to hormone-independent, they are associated with increased growth factor signaling, enhanced cellular motility, and the epithelial to mesenchymal transition (EMT). Given the complexity of cancer stem cells (CSC) and their implications on endocrine resistance and EMT, we sought to understand their contribution towards the development of aromatase inhibitor resistant breast cancer. Cells cultured three dimensionally as mammospheres are enriched for CSCs and more accurately recapitulates tumors in vivo. Therefore, a global proteomic analysis was conducted using letrozole resistant breast cancer cells (LTLT-Ca) mammospheres and compared to their adherent counterparts. Results demonstrated over 1000 proteins with quantitative abundance ratios were identified. Among the quantified proteins, 359 were significantly altered (p < 0.05), where 173 were upregulated and 186 downregulated (p < 0.05, fold change >1.20). Notably, midasin, a chaperone protein required for maturation and nuclear export of the pre-60S ribosome was increased 35-fold. Protein expression analyses confirmed midasin is ubiquitously expressed in normal tissue but is overexpressed in lobular and ductal breast carcinoma tissue as well as ER+ and ER- breast cancer cell lines. Functional enrichment analyses indicated that 19 gene ontology terms and one KEGG pathway were over-represented by the down-regulated proteins and both were associated with protein synthesis. Increased midasin was strongly correlated with decreased relapse free survival in hormone independent breast cancer. For the first time, we characterized the global proteomic signature of CSC-enriched letrozole-resistant cells associated with protein synthesis, which may implicate a role for midasin in endocrine resistance.

Metformin Inhibits Migration and Invasion by Suppressing ROS Production and COX2 Expression in MDA-MB-231 Breast Cancer Cells.

BACKGROUND: Several mechanisms of action have been proposed to explain the apparent antineoplastic functions of metformin, many of which are observed at high concentrations that may not be reflective of achievable tissue concentrations. We propose that metformin at low concentrations functions to inhibit ROS production and inflammatory signaling in breast cancer, thereby reducing metastasis. METHODS: Using the highly invasive MDA-MB-231 breast carcinoma model, we ascertained the impact of metformin on cell viability by DNA content analysis and fluorescent dye exclusion. Migration and invasion assays were performed using a modified Boyden chamber assay and metastasis was ascertained using the chorioallantoic membrane (CAM) assay. PGE2 production was measured by Enzyme-Linked Immunosorbent Assay (ELISA). COX2 and ICAM1 levels were determined by flow cytometry immunoassay. RESULTS: Metformin acutely decreased cell viability and caused G2 cell cycle arrest only at high concentrations (10 mM). At 100 µM, however, metformin reduced ICAM1 and COX2 expression, as well as reduced PGE2 production and endogenous mitochondrial ROS production while failing to significantly impact cell viability. Consequently, metformin inhibited migration, invasion in vitro and PGE2-dependent metastasis in CAM assays. CONCLUSION: At pharmacologically achievable concentrations, metformin does not drastically impact cell viability, but inhibits inflammatory signaling and metastatic progression in breast cancer cells.

Glyceollin I Reverses Epithelial to Mesenchymal Transition in Letrozole Resistant Breast Cancer through ZEB1.

Although aromatase inhibitors are standard endocrine therapy for postmenopausal women with early-stage metastatic estrogen-dependent breast cancer, they are limited by the development of drug resistance. A better understanding of this process is critical towards designing novel strategies for disease management. Previously, we demonstrated a global proteomic signature of letrozole-resistance associated with hormone-independence, enhanced cell motility and implications of epithelial mesenchymal transition (EMT). Letrozole-resistant breast cancer cells (LTLT-Ca) were treated with a novel phytoalexin, glyceollin I, and exhibited morphological characteristics synonymous with an epithelial phenotype and decreased proliferation. Letrozole-resistance increased Zinc Finger E-Box Binding Homeobox 1 (ZEB1) expression (4.51-fold), while glyceollin I treatment caused a -3.39-fold reduction. Immunofluorescence analyses resulted of glyceollin I-induced increase and decrease in E-cadherin and ZEB1, respectively. In vivo studies performed in ovariectomized, female nude mice indicated that glyceollin treated tumors stained weakly for ZEB1 and N-cadherin and strongly for E-cadherin. Compared to letrozole-sensitive cells, LTLT-Ca cells displayed enhanced motility, however in the presence of glyceollin I, exhibited a 68% and 83% decrease in invasion and migration, respectively. These effects of glyceollin I were mediated in part by inhibition of ZEB1, thus indicating therapeutic potential of glyceollin I in targeting EMT in letrozole resistant breast cancer.

Proteomic signatures of acquired letrozole resistance in breast cancer: suppressed estrogen signaling and increased cell motility and invasiveness.

Aromatase inhibitors, such as letrozole, have become the first-line treatment for postmenopausal women with estrogen-dependent breast cancer. However, acquired resistance remains a major clinical obstacle. Previous studies demonstrated constitutive activation of the MAPK signaling, overexpression of HER2, and down-regulation of aromatase and ERα in letrozole-resistant breast cancer cells. Given the complex signaling network involved in letrozole-refractory breast cancer and the lack of effective treatment for hormone resistance, further investigation of aromatase inhibitor resistance by a novel systems biology approach may reveal previously unconsidered molecular changes that could be utilized as therapeutic targets. This study was undertaken to characterize for the first time global proteomic alterations occurring in a letrozole-resistant cell line. A quantitative proteomic analysis of the whole cell lysates of LTLT-Ca (resistant) versus AC-1 cells (sensitive) was performed to identify significant protein expression changes. A total of 1743 proteins were identified and quantified, of which 411 were significantly up-regulated and 452 significantly down-regulated (p < 0.05, fold change > 1.20). Bioinformatics analysis revealed that acquired letrozole resistance is associated with a hormone-independent, more aggressive phenotype. LTLT-Ca cells exhibited 84% and 138% increase in migration and invasion compared with the control cells. The ROCK inhibitor partially abrogated the enhanced migration and invasion of the letrozole-resistant cells. Flow cytometric analyses also demonstrated an increase in vimentin and twist expression in letrozole-resistance cells, suggesting an onset of epithelial to mesenchymal transition (EMT). Moreover, targeted gene expression arrays confirmed a 28-fold and sixfold up-regulation of EGFR and HER2, respectively, whereas ERα and pS2 were dramatically reduced by 28-fold and 1100-fold, respectively. Taken together, our study revealed global proteomic signatures of a letrozole-resistant cell line associated with hormone independence, enhanced cell motility, EMT and the potential values of several altered proteins as novel prognostic markers or therapeutic targets for letrozole resistant breast cancer.

Dichotomous roles for the orphan nuclear receptor NURR1 in breast cancer.

BACKGROUND: NR4A orphan nuclear receptors are involved in multiple biological processes which are important in tumorigenesis such as cell proliferation, apoptosis, differentiation, and glucose utilization. The significance of NR4A family member NURR1 (NR4A2) in breast cancer etiology has not been elucidated. The purpose of this study was to ascertain the impact of NURR1 expression on breast transformation, tumor growth, and breast cancer patient survival. METHODS: We determined the expression of NURR1 in normal breast versus breast carcinoma in tissue microarrays (immunohistochemistry), tissue lysates (immunoblot), and at the mRNA level (publically available breast microarrays). In addition NURR1 expression was compared among breast cancer patients in cohorts based on p53 expression, estrogen receptor α expression, tumor grade, and lymph node metastases. Kaplan-Meier survival plots were used to determine the correlation between NURR1 expression and relapse free survival (RFS). Using shRNA-mediated silencing, we determined the effect of NURR1 expression on tumor growth in mouse xenografts. RESULTS: Results from breast cancer tissue arrays demonstrate a higher NURR1 expression in the normal breast epithelium compared to breast carcinoma cells (p ≤ 0.05). Among cases of breast cancer, NURR1 expression in the primary tumors was inversely correlated with lymph node metastases (p ≤ 0.05) and p53 expression (p ≤ 0.05). Clinical stage and histological grade were not associated with variation in NURR1 expression. In gene microarrays, 4 of 5 datasets showed stronger mean expression of NURR1 in normal breast as compared to transformed breast. Additionally, NURR1 expression was strongly correlated with increase relapse free survival (HR = 0.7) in a cohort of all breast cancer patients, but showed no significant difference in survival when compared among patients whom have not been treated systemically (HR = 0.91). Paradoxically, NURR1 silenced breast xenografts showed significantly decreased growth in comparison to control, underscoring a biphasic role for NURR1 in breast cancer progression. CONCLUSIONS: NURR1 function presents a dichotomy in breast cancer etiology, in which NURR1 expression is associated with normal breast epithelial differentiation and efficacy of systemic cancer therapy, but silencing of which attenuates tumor growth. This provides a strong rationale for the potential implementation of NURR1 as a pharmacologic target and biomarker for therapeutic efficacy in breast cancer.

Metformin induces a senescence-associated gene signature in breast cancer cells.

Diabetic patients taking metformin have lower incidence of breast cancer than those taking other anti-diabetic medications. Additionally, triple negative breast cancer (TNBC), a form of breast cancer disproportionately afflicting premenopausal African American women, shows atypical susceptibility to metformin's antiproliferative effect. The mechanisms involved in metformin's function in TNBC has not yet been fully elucidated. Therefore, we sought to identify pathways regulated by metformin in using the MDA-MB-468 TNBC cell model. Metformin dose-dependently caused apoptosis, decreased cell viability, and induced cell morphology/chromatin condensation consistent with the permanent proliferative arrest. Furthermore, gene expression arrays revealed that metformin caused expression of stress markers DDIT3, CYP1A1,and GDF-15 and a concomitant reduction in PTGS1 expression. Our findings show that metformin may affect the viability and proliferative capacity of TNBC by inducing an antiproliferative gene signature, and that metformin may be effective in the treatment/prevention of TNBC.

Surface modification of poly(methyl methacrylate) microfluidic devices for high-resolution separations of single-stranded DNA.

While polymer-based microfluidic devices offer some unique opportunities in developing low-cost systems for a variety of application areas, the ability to sort electrophoretically with high efficiency a number of different targets has remained somewhat elusive with an example consisting of achieving single base resolution as required for DNA sequencing. While the reasons for this are many-fold, it is clear that some type of coating is required on the polymer substrate to suppress the EOF and/or minimize potential solute/wall interactions. To this end, we report on a simple grafting procedure to allow the formation of polymer coats, which in this example used linear polyarcylamides (LPAs), onto a poly(methyl methacrylate) (PMMA) microfluidic device. The procedure involved creating an amine-terminated PMMA surface by appropriately functionalizing the PMMA through either a chemical or photochemical process. The aminated surface could then be used to covalently anchor methacrylic acid, which was used as a scaffold to produce LPAs on the surface through radical polymerization of acrylamide. The resulting surfaces demonstrated EOFs that were nearly an order of magnitude smaller than native PMMA. In addition, these LPA-coated devices could produce highly reproducible migration times of over approximately 20 runs with plate numbers exceeding 10(5) m(-1). Using gel electrophoretic analysis of a single base track generated from an M13mp18 template using Sanger cycle sequencing and dye-primer chemistry, the resolution value obtained for bases 199 and 200 was 0.18 while for bases 208 and 209 it was 0.21. For the native PMMA, these bands were found to comigrate.

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips.

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of approximately 7.6 +/- 1.6 microg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 +/- 5%. The immobilization and purification assay using this PPC microchip could be performed within approximately 25 min as follows: (i) DNA immobilization approximately 6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption approximately 6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for 'reactivation' of the PC surface with UV light.

Near-infrared time-resolved fluorescence lifetime determinations in poly(methylmethacrylate) microchip electrophoresis devices.

High aspect-ratio microstructures were hot-embossed in polymer substrates with a molding tool fabricated using lithography/electroplating/forming (LIGA). The resulting devices were used for the electrophoretic separation of oligonucleotides labeled with near-infrared (near-IR) dyes. Near-IR time-resolved fluorescence was used as an identification method for the labeling dyes. The detection apparatus consisted of a pulsed laser diode operating at 680 nm, a single-photon avalanche diode, an integrated microscope, and a PC-board incorporating time-correlated single photon counting electronics. Investigation of the optical quality and amount of autofluorescence generated from different polymer substrates was carried out in the near-IR region for determining compatibility with time-resolved fluorescence. Our results revealed that of several poly(methylmethacrylate)(PMMA) substrates, brand Plexiglas offered minimal replication errors in the embossed features using appropriate embossing conditions with low background fluorescence contributions to the observed decay. Near-IR dye-labeled oligonucleotides were separated to determine the applicability of fluorescence lifetime discrimination between Cy5.5 (tauf = 930 ps) and IRD700 (tauf = 851 ps) labeling dyes during the microchip separation. These dyes were used to label T-fragments (thymine) of an M13mp18 ssDNA template. The DNA ladders were electrophoresed at 130 V/cm in a 4% linear polyacrylamide gel (LPA) gel matrix in a 9.5 cm long serpentine channel heated to 50 degrees C. The electropherogram revealed that the lifetimes could be accurately read well beyond 450 bases, although single-base pair resolution in the electropherogram was difficult to achieve due to potential solute-wall interactions in the polymer microdevice or the electroosmotic flow (EOF) properties of the device. The relative standard deviations secured for individual bands in the electropherogram were similar to those obtained using capillary gel electrophoresis, in spite of the lower load volume.

Contact conductivity detection in poly(methyl methacrylate)-based microfluidic devices for analysis of mono- and polyanionic molecules.

An on-column contact conductivity detector was developed for the analysis of various mono- and polyanionic compounds separated by electrophoresis chips fabricated in poly(methyl methacrylate) (PMMA) using hot embossing techniques from Ni electroforms. The detector consisted of a pair of Pt wires (127 microm diameter) with an end-to-end spacing of approximately 20 microm and situated within the fluidic channel. The waveform applied to the electrode pair was a bipolar pulse with a frequency of 5.0 kHz and was used to reduce the charging current from measurement so that the current recorded at the end of one pulse is more representative of the solution conductivity. Using the detector, separations of amino acids, peptides, proteins, and oligonucleotides were demonstrated. For the amino acids and peptides, free-solution zone electrophoresis was performed. A calibration plot for the amino acid alanine was found to be linear from approximately 10 to 100 nM in a carrier electrolyte consisting of 10 mM triethylamonium acetate. The concentration detection limit was found to be 8.0 nM, with the corresponding mass detection limit equal to 3.4 amol (injection volume = 425 pL). The protein separations with conductivity detection were performed using MEKC, in which the carrier electrolyte contained the anionic surfactant sodium dodecyl sulfate (SDS) above its cmc. Near baseline resolution was achieved in the PMMA microchip for a solution containing 8 different proteins. In the case of the DNA fragments, capillary electrochromatography was used with a C18-modified PMMA chip and a carrier electrolyte containing an ion-pairing agent.