Disruption of apoptosis may allow metastatic cell survival and confer resistance to chemotherapeutic drugs. We have analysed the molecular pathways that activate these survival genes in specific sites of metastasis. Estrogen receptor-negative breast cancer cell line MDA-MB435 and two metastatic sublines derived from lung (435L) and brain (435B) were analysed for the expression of members of the Bcl-2 family of apoptosis regulators. The levels of Bcl-2 were higher in the metastatic sublines than in parental cells, which correlated with the activation of Stat3, but not with the expression and/or activation of known bcl-2 transcription factors (CREB and WT1). In the brain subline, both expression of Bcl-2 and Stat3 activation were induced by epidermal growth factor and abrogated after treatment with kinase inhibitors specific for epidermal growth factor receptor or Jak2. Furthermore, transfection of 435B with a dominant-negative Stat3 markedly reduced the expression of Bcl-2 protein, whereas transient expression of a constitutively active Stat3 increased Bcl-2 in parental 435 cells. In addition, blockade of Stat3 activation by treatment with epidermal growth factor receptor and Jak2 kinase inhibitors or transfection with a dominant negative Stat3, sensitizes 435B cells to chemotherapy-induced apoptosis. Our data suggest that an increased activation of the Stat3 -Bcl-2 pathway in estrogen receptor-negative metastatic breast cancer cell lines confer a survival advantage to these cells and contribute to their chemoresistance.

The lungs are a frequent target of metastatic breast cancer cells, but the underlying molecular mechanisms are unclear. All existing data were obtained either using statistical association between gene expression measurements found in primary tumors and clinical outcome, or using experimentally derived signatures from mouse tumor models. Here, we describe a distinct approach that consists of using tissue surgically resected from lung metastatic lesions and comparing their gene expression profiles with those from nonpulmonary sites, all coming from breast cancer patients. We show that the gene expression profiles of organ-specific metastatic lesions can be used to predict lung metastasis in breast cancer. We identified a set of 21 lung metastasisassociated genes. Using a cohort of 72 lymph node-negative breast cancer patients, we developed a 6-gene prognostic classifier that discriminated breast primary cancers with a significantly higher risk of lung metastasis. We then validated the predictive ability of the 6-gene signature in 3 independent cohorts of breast cancers consisting of a total of 721 patients. Finally, we show that the signature improves risk stratification independently of known standard clinical variables and a previously established lung metastasis signature based on an experimental breast cancer metastasis model. [Cancer Res 2008;68(15):6092-9]

Although molecular classification brings interesting insights into breast cancer taxonomy, its implementation in daily clinical care is questionable because of its expense and the information supplied in a single sample allocation is not sufficiently reliable. New approaches, based on a panel of small molecules derived from the global or targeted analysis of metabolic profiles of cells, have found a correlation between activation of de novo lipogenesis and poorer prognosis and shorter disease-free survival for many tumors. We hypothesized that the lipid content of breast cancer cells might be a useful indirect measure of a variety of functions coupled to breast cancer progression. Raman microspectroscopy was used to characterize metabolism of breast cancer cells with different degrees of malignancy. Raman spectra from MDA-MB-435, MDA-MB-468, MDA-MB-231, SKBR3, MCF7 and MCF10A cells were acquired with an InVia Raman microscope (Renishaw) with a backscattered configuration. We used Principal Component Analysis and Partial Least Squares Discriminant Analyses to assess the different profiling of the lipid composition of breast cancer cells. Characteristic bands related to lipid content were found at 3014, 2935, 2890 and 2845 cm−1, and related to lipid and protein content at 2940 cm−1. A classificatory model was generated which segregated metastatic cells and non-metastatic cells without basal-like phenotype with a sensitivity of 90% and a specificity of 82.1%. Moreover, expression of SREBP-1c and ABCA1 genes validated the assignation of the lipid phenotype of breast cancer cells. Indeed, changes in fatty acid unsaturation were related with the epithelial-to-mesenchymal transition phenotype. Raman microspectroscopy is a promising technique for characterizing and classifying the malignant phenotype of breast cancer cells on the basis of their lipid profiling. The algorithm for the discrimination of metastatic ability is a first step towards stratifying breast cancer cells using this rapid and reagent-free tool.

Metastasis is a highly complex process involving the survival of tumor cells, both in the blood stream and within specific organs. Cell-death and survival are determined by a number of gene products from an expanding family of the Bcl-2 gene, either promoting or preventing apoptosis. Furthermore, the survival of tumor cells may favor the accumulation of additional genetic alterations causing further growth and invasive opportunities which may lead to metastasis. To examine whether the prevention of cell-death influences the metastatic behavior, we transfected a human breast cancer cell line MDA-MB-435 with the Bcl-x L cDNA and then studied metastatic ability of the selected clones in vivo. Our results show that Bcl-x L -clones had a decreased tumor growth latency and an increased metastatic ability. Apoptosisresistance to cytokines was induced in 435 cells by Bcl-x Lexpression with minor modifications in their proliferation rates. These cells also showed diminished adhesion to extracellular matrix proteins and a survival advantage in suspension over 435/Neo cells. Moreover, to determine survival in blood stream and in cells lodged in the lungs, we injected 435/Bcl-x L and 435/Neo cells at 1 : 3 proportion i.v., and animals were killed at intervals of 15' to 16 h after injection. Tumor cells were recovered from the lungs and Southern-blot analysis revealed the presence of exogenous Bcl-x L cDNA. These results showed that 435/Bcl-x L cells had a survival advantage in circulation over 435/Neo cells. This advantage in vivo was attributable to Bcl-x L expression. We conclude that Bcl-x L expression in breast cancer cells can increase metastatic activity. This advantage could be created by inducing resistance to apoptosis against cytokines, increasing cell survival in circulation, and enhancing anchorageindependent growth.Cell Death and Differentiation (2000) 7, 350 ± 359.

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