Introduction: A subpopulation (CD44⁺/CD24^-) of breast cancer cells has been reported to have stem/progenitor cell properties. The aim of this study was to investigate whether this subpopulation of cancer cells has the unique ability to invade, home, and proliferate at sites of metastasis. Methods: CD44 and CD24 expression was determined by flow cytometry. Northern blotting was used to determine the expression of proinvasive and 'bone and lung metastasis signature' genes. A matrigel invasion assay and intracardiac inoculation into nude mice were used to evaluate invasion, and homing and proliferation at sites of metastasis, respectively. Results: Five among 13 breast cancer cell lines examined (MDA-MB-231, MDA-MB-436, Hs578T, SUM1315, and HBL-100) contained a higher percentage (>30%) of CD44⁺/CD24^- cells. Cell lines with high CD44⁺/CD24^- cell numbers express basal/mesenchymal or myoepithelial but not luminal markers. Expression levels of proinvasive genes (IL-1α, IL-6, IL-8, and urokinase plasminogen activator [UPA]) were higher in cell lines with a significant CD44⁺/CD24^- population than in other cell lines. Among the CD44⁺/CD24^--positive cell lines, MDA-MB-231 has the unique property of expressing a broad range of genes that favor bone and lung metastasis. Consistent with previous studies in nude mice, cell lines with CD44⁺/CD24^- subpopulation were more invasive than other cell lines. However, only a subset of CD44⁺/CD24^--positive cell lines was able to home and proliferate in lungs. Conclusion: Breast cancer cells with CD44⁺/CD24^- subpopulation express higher levels of proinvasive genes and have highly invasive properties. However, this phenotype is not sufficient to predict capacity for pulmonary metastasis.

Estrogen receptors (ERs) mediate most of the biological effects of estrogen in mammary and uterine epithelial cells by binding to estrogen response elements in the promoter region of target genes or through protein-protein interactions. Anti-estrogens such as tamoxifen inhibit the growth of ER-positive breast cancers by reducing the expression of estrogen-regulated genes. However, anti-estrogen-resistant growth of ER-positive tumors remains a significant clinical problem. Here we show that phosphatidylinositol (PI) 3-kinase and AKT activate ERα in the absence of estrogen. Although PI 3-kinase increased the activity of both estrogen-independent activation function 1 (AF-1) and estrogen-dependent activation function 2 (AF-2) of ERα, AKT increased the activity of only AF-1. PTEN and a catalytically inactive AKT decreased PI 3-kinase-induced AF-1 activity, suggesting that PI 3-kinase utilizes AKT-dependent and AKT-independent pathways in activating ERα. The consensus AKT phosphorylation site Ser-167 of ERα is required for phosphorylation and activation by AKT. In addition, LY294002, a specific inhibitor of the PI 3-kinase/AKT pathway, reduced phosphorylation of ERα in vivo. Moreover, AKT overexpression led to up-regulation of estrogen-regulated pS2 gene, Bcl-2, and macrophage inhibitory cytokine 1. We demonstrate that AKT protects breast cancer cells from tamoxifen-induced apoptosis. Taken together, these results define a molecular link between activation of the PI 3-kinase/AKT survival pathways, hormone-independent activation of ERα, and inhibition of tamoxifen-induced apoptotic regression.

Metastasis of cancer cells is a complex process involving multiple steps including invasion, angiogenesis, and trafficking of cancer cells through blood vessels, extravasations, organ-specific homing, and growth. While matrix metalloproteinases, urokinase-type plasminogen activator, and cytokines play a major role in invasion and angiogenesis, chemokines such as stromal derived factor-1α (SDF-1α) and their receptors such as CXCR4 are thought to play a critical role in motility, homing, and proliferation of cancer cells at specific metastatic sites. We and others have previously reported that the extracellular signal-activated transcription factor NF-κB up-regulates the expression of matrix metalloproteinases, urokinase-type plasminogen activator, and cytokines in highly metastatic breast cancer cell lines. In this report, we demonstrate that NF-κB regulates the motility of breast cancer cells by directly up-regulating the expression of CXCR4. Overexpression of the inhibitor of κB (IκB) in breast cancer cells with constitutive NF-κB activity resulted in reduced expression of CXCR4 and a corresponding loss of SDF-1α-mediated migration in vitro. Introduction of CXCR4 cDNA into IκB-expressing cells restored SDF-1α-mediated migration. Electrophoretic mobility shift assays and transient transfection assays revealed that the NF-κB subunits p65 and p50 bind directly to sequences within the –66 to +7 region of the CXCR4 promoter and activate transcription. We also show that the cell surface expression of CXCR4 and the SDF-1α-mediated migration are enhanced in breast cancer cells isolated from mammary fat pad xenografts compared with parental cells grown in culture. A further increase in CXCR4 cell surface expression and SDF-1α-mediated migration was observed with cancer cells that metastasized to the lungs. Taken together, these results implicate NF-κB in the migration and the organ-specific homing of metastatic breast cancer cells.

The transcription factor nuclear factor kappa B (NF-κB) is constitutively active in both cancer cells and stromal cells of breast cancer; however, the precise role of activated NF-κB in cancer progression is not known. Using parental MCF10A cells and a variant that expresses the myoepithelial marker p63 stably overexpressing the constitutively active p65 subunit of NF-κB (MCF10A/p65), we show that NF-κB suppresses the expression of epithelial specific genes E-cadherin and desmoplakin and induces the expression of the mesenchymal specific gene vimentin. P65 also suppressed the expression of p63 and the putative breast epithelial progenitor marker cytokeratin 5/6. MCF10A/p65 cells were phenotypically similar to cells undergoing epithelial to mesenchymal transition (EMT). MCF10A/p65 cells failed to form characteristic acini in three-dimensional Matrigel. Analysis of parental and MCF10A/p65 cells for genes previously shown to be involved in EMT revealed elevated expression of ZEB-1 and ZEB-2 in MCF10A/p65 cells compared to parental cells. In transient transfection assays, p65 increased ZEB-1 promoter activity. Furthermore, MCF10A cells overexpressing ZEB-1 showed reduced E-cadherin and p63 expression and displayed an EMT phenotype. The siRNA against ZEB-1 or ZEB-2 reduced the number of viable MCF10A/p65 but not parental cells, suggesting the dependence of MCF10A/p65 cells to ZEB-1 and ZEB-2 for cell cycle progression or survival. MCF10A cells chronically exposed to tumor necrosis factor alpha (TNFα), a potent NF-κB inducer, also exhibited the EMT-like phenotype and ZEB-1/ZEB-2 induction, both of which were reversed following TNFα withdrawal.

We recently published PROGgene, a tool that can be used to study prognostic implications of genes in various cancers. The first version of the tool had several areas for improvement. In this paper we present some major enhancements we have made on the existing tool in the new version, PROGgeneV2. In PROGgeneV2, we have made several modifications to enhance survival analysis capability of the tool. First, we have increased the repository of public studies catalogued in our tool by almost two folds. We have also added additional functionalities to perform survival analysis in a variety of new ways. Survival analysis can now be performed on a) single genes b) multiple genes as a signature, c) ratio of expression of two genes, and d) curated/published gene signatures in new version. Users can now also adjust the survival analysis models for available covariates. Users can study prognostic implications of entire gene signatures in different cancer types, which are searchable by keywords. Also, unique to our tool, in the new version, users will be able to upload and use their own datasets to perform survival analysis on genes of interest. We believe, like its predecessor, PROGGeneV2 will continue to be useful for the scientific community for formulating research hypotheses and designing mechanistic studies. With added datasets PROGgeneV2 is the most comprehensive survival analysis tool available. PROGgeneV2 is available at http://www.compbio.iupui.edu/proggene .