Parental Breast Cancer Cells Research Articles

Abstract 2844: Systems biology analysis of tumor and stromal genes in different metastatic microenvironments

Abstract Metastasis is a multistage process that requires cancer cells to escape from the primary tumor, survive in the circulation, seed at distant sites and colonize these foreign tissue environments. Each of these processes involves rate-limiting steps that are influenced by stromal cells of the tumor microenvironment. Our goal is to dissect organ-site specific signatures of tumor-stroma interactions in metastasis. In this approach we devised an experimental and computational strategy to enable the simultaneous analysis of tumor and stromal genes in lesions from three distinct metastatic microenvironments. We took advantage of a recently developed system where organ-site specific metastatic variants were selected in vivo from a parental breast cancer cell line and home to the brain, the bone and the lung in xenografted animals. An important technological advance is the HuMu ProtIn (Protease/Inhibitor) array that was designed to distinguish between human (Hu) and mouse (Mu) gene expression. By using the HuMu ProtIn array we can determine whether differentially expressed proteases and endogenous protease inhibitors are of tumor (human) or stromal (mouse) origin. This allowed us to simultaneously capture the tumor and stromal contributions in intact tumors. After profiling of brain, bone and lung metastases at different stages of development we employed knock-down and knock-out strategies to experimentally validate the biological relevance of predicted candidate genes including the serine protease Htra1 and the cysteine protease cathepsin S. Cathepsin S represents an interesting example as the expression of tumor-derived cathepsin S decreases during the progression from early to late brain metastasis, whereas the expression of stromal-derived cathepsin S increases over time. Targeting either tumor- or stromal derived cathepsin S did not significantly prolong tumor-free survival, however the combined ablation of tumor- and stromal derived cathepsin S resulted in a dramatic impairment of brain metastasis formation. This study emphasizes the need for a better understanding of the complex reciprocal interactions between tumor cells and stromal cells with the ultimate goal to improve the predictive value of prognostic evaluation and to develop combination therapies that target the tumor along with its microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2844. doi:10.1158/1538-7445.AM2011-2844

Abstract LB-261: Cancer stem cell characteristics exhibited by doxorubicin-selected drug-resistant breast cancer cells

Abstract Background: Cancer stem cells (CSCs) are presumed to possess the virtually unlimited ability to proliferate while preserving self-renewal. These cells are also highly resistant to chemotherapeutics, a feature which correlates with overexpression of ATP-binding cassette (ABC) transporters. No reports to our knowledge have shown that prolonged continuous selection of cells for drug resistance enriches cancer stem-like cells. Methods: We examined parental and drug-selected breast cancer cell lines to determine whether there is a relationship between drug resistance and the presence of cells with a CSC phenotype. MCF-7/ADR cells, generated from weakly tumorigenic parental MCF7 cells by continuous exposure to increasing concentrations of doxorubicin, were examined by microarray analysis, flow cytometry, invasion, in vivo tumorigenicity and other CSC characteristics. The putative CSC population was also examined for expression of multidrug resistance (MDR) genes using TaqMan Low Density Arrays. Results: Pathway analysis showed that MCF-7/ADR cells express ABCB1 as well as genes with patterns similar to breast CSCs. MCF-7/ADR cells grow in 3D cultures as mammospheres, are highly invasive and tumorigenic. In contrast to parental cells, over 30% of MCF-7/ADR cells are CD44+/CD24-. The CD44+/CD24- cells have the ability to self-renew and overexpress many MDR genes when compared to the CD44+/CD24+ population. No enrichment in CD44+/CD24- or CD133+ populations was detected in MCF-7/MDR or other cells with forced expression of the ABCB1 transporter or MDR genes. Conclusions: Cells with characteristics of CSCs increased in number following selection for doxorubicin resistance. Based on our findings, we speculate that prolonged drug treatment in vivo may result in expansion of a CSC-like population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-261.

Proteomic Identification of Putative Biomarkers of Radiotherapy Resistance: A Possible Role for the 26S Proteasome?

We aimed to identify putative predictive protein biomarkers of radioresistance. Three breast cancer cell lines (MCF7, MDA-MB-231, and T47D) were used as in vitro models to study radioresistance. Inherent radiosensitivities were examined using a clonogenic survival assay. It was revealed that each cell line differed in their response to radiotherapy. These parental breast cancer cell lines were used to establish novel derivatives (MCF7RR, MDA-MB-231RR, and T47DRR) displaying significant resistance to ionizing radiation. Derivative cells were compared with parental cells to identify putative biomarkers associated with the radioresistant phenotype. To identify these biomarkers, complementary proteomic screening approaches were exploited encompassing two-dimensional gel electrophoresis in combination with mass spectrometry, liquid chromatography coupled with tandem mass spectrometry and quantitative proteomics using iTRAQ technology. A large number of potential biomarkers were identified, and several of these were confirmed using Western blot analysis. In particular, a decrease in the expression of the 26S proteasome was found in all radioresistant derivatives when compared with the respective parent cells. Decreased expression of this target was also found to be associated with radioresistant laryngeal tumors (P = .05) in a small pilot immunohistochemical study. These findings suggest that the 26S proteasome may provide a general predictive biomarker for radiotherapy outcome.

Loss of α-Catenin Decreases the Strength of Single E-cadherin Bonds between Human Cancer Cells

The progression of several human cancers correlates with the loss of cytoplasmic protein alpha-catenin from E-cadherin-rich intercellular junctions and loss of adhesion. However, the potential role of alpha-catenin in directly modulating the adhesive function of individual E-cadherin molecules in human cancer is unknown. Here we use single-molecule force spectroscopy to probe the tensile strength, unstressed bond lifetime, and interaction energy between E-cadherins expressed on the surface of live human parental breast cancer cells lacking alpha-catenin and these cells where alpha-catenin is re-expressed. We find that the tensile strength and the lifetime of single E-cadherin/E-cadherin bonds between parental cells are significantly lower over a wide range of loading rates. Statistical analysis of the force displacement spectra reveals that single cadherin bonds between cancer cells feature an exceedingly low energy barrier against tensile forces and low molecular stiffness. Disassembly of filamentous actin using latrunculin B has no significant effect on the strength of single intercellular E-cadherin bonds. The absence of alpha-catenin causes a dominant negative effect on both global cell-cell adhesion and single E-cadherin bond strength. These results suggest that the loss of alpha-catenin alone drastically reduces the adhesive force between individual cadherin pairs on adjoining cells, explain the global loss of cell adhesion in human breast cancer cells, and show that the forced expression of alpha-catenin in cancer cells can restore both higher intercellular avidity and intercellular E-cadherin bond strength.

Microarray analysis reveals potential mechanisms of BRMS1-mediated metastasis suppression

We used Affymetrix microarrays to compare gene expression profiles of the metastatic parental breast cancer cell line MDA-MB-435 (435) and the non-metastatic daughter cell line created by the stable expression of the BReast cancer Metastasis Suppressor 1 (BRMS1) gene in 435 cells, MDA-MB-435-BRMS1 (435/BRMS1). Analysis of microarray data provided insight into some of the potential mechanisms by which BRMS1 inhibits tumor formation at secondary sites. Furthermore, due to the importance of the microenvironment, we also examined gene expression under different growth conditions (i.e., plus or minus serum). Expression of 565 genes was significantly (adjusted P-value <0.05) altered regardless of in vitro growth conditions. BRMS1 expression significantly increased multiple major histocompatability complex (MHC) genes and significantly decreased expression of several genes associated with protein localization and secretion. The pattern of gene expression associated with BRMS1 expression suggests that metastasis suppression may be mediated by enhanced immune recognition, altered transport, and/or secretion of metastasis-associated proteins.

Parallel microfluidic networks for studying cellular response to chemical modulation

A microfluidic chip featuring parallel gradient-generating networks etched on glass plate was designed and fabricated. The dam and weir structures were fabricated to facilitate cell positioning and seeding, respectively. The microchip contains five gradient generators and 30 cell chambers where the resulted concentration gradients of drugs are delivered to stimulate the on-chip cultured cells. This microfluidics exploits the advantage of lab-on-a-chip technology by integrating the generation of drug concentration gradients and a series of cell operations including seeding, culture, stimulation and staining into a chip. Steady parallel concentration gradients were generated by flowing two fluids in each network. The microchip described above was applied in studying the role of reduced glutathione (GSH) in MCF-7 cells’ chemotherapy sensitivity. The parental breast cancer cell line, MCF-7 and the derived adriamycin resistant cell line MCF-7 adm were treated with concentration gradients of arsenic trioxide (ATO) and N-acetyl cysteine (NAC) for GSH modulation, followed by exposure to adriamycin. The intracellular GSH level and cell viability were assessed by fluorescence image analysis. GSH levels of both cell lines were down-regulated upon ATO treatment and up-regulated upon NAC treatment. For both cell lines, suppression of intracellular GSH by treatment with ATO has been shown to increase chemotherapy sensitivity; conversely, elevation of intracellular GSH by treatment with NAC leads to increased drug resistance. The results indicated that high intracellular GSH level has negative effect on chemotherapy sensitivity, while depletion of cellular GSH may serve as an effective way to improve chemotherapy sensitivity. The integrated microfluidic chip is able to perform multiparametric pharmacological profiling with easy operation, thus, holds great potential for extrapolation to the high-content drug screening.