Nontransgenic models of breast cancer

Numerous models have been developed to address key elements in the biology of breast cancer development and progression. No model is ideal, but the most useful are those that reflect the natural history and histopathology of human disease, and allow for basic investigations into underlying cellular and molecular mechanisms. We describe two types of models: those that are directed toward early events in breast cancer development (hyperplastic alveolar nodules [HAN] murine model, MCF10AT human xenograft model); and those that seek to reflect the spectrum of metastatic disease (murine sister cell lines 67, 168, 4T07, 4T1). Collectively, these models provide cell lines that represent all of the sequential stages of progression in breast disease, which can be modified to test the effect of genetic changes

Central carbon metabolism in the progression of mammary carcinoma

There is a growing belief that the metabolic program of breast tumor cells could be a therapeutic target. Yet, without detailed information on central carbon metabolism in breast tumors it is impossible to know which metabolic pathways to target, and how their inhibition might influence different stages of breast tumor progression. Here we perform the first comprehensive profiling of central metabolism in the MCF10 model of mammary carcinoma, where the steps of breast tumor progression (transformation, tumorigenicity and metastasis) can all be examined in the context of the same genetic background. The metabolism of [U-13C]-glucose by a series of progressively more aggressive MCF10 cell lines was tracked by 2D NMR and mass spectrometry. From this analysis the flux of carbon through distinct metabolic reactions was quantified by isotopomer modeling. The results indicate widespread changes to central metabolism upon cellular transformation including increased carbon flux through the pentose phosphate pathway (PPP), the TCA cycle, as well as increased synthesis of glutamate, glutathione and fatty acids (including elongation and desaturation). The de novo synthesis of glycine increased upon transformation as well as at each subsequent step of breast tumor cell progression. Interestingly, the major metabolic shift in metastatic cells is a large increase in the de novo synthesis of proline. This work provides the first comprehensive view of changes to central metabolism as a result of breast tumor progression

Smad4-expression is decreased in breast cancer tissues: a retrospective study

BACKGROUND: Although transforming growth factor β (TGF-β) typically inhibits proliferation of epithelial cells, consistent with a tumor suppressor activity, it paradoxically also exhibits pro-metastatic activity in the later stages of carcinogenesis. Since tumors often display altered TGF-β signaling, particularly involving the Smad-pathway, we investigated the role of Smad4-expression in breast cancer. METHODS: Smad4 expression was investigated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue from 197 samples of primary breast cancer obtained between 1986 and 1998. The prognostic value of Smad4-expression was analyzed. RESULTS: Smad4 expression was found to be reduced in lobular and ductal breast carcinoma as compared to surrounding uninvolved lobular and ductal breast epithelia (p < 0.001, n = 50). Smad4-expression correlated positively with expression of TGF-β-receptor I (p < 0.001, n = 197) and TGF-β-receptor II (p < 0.001, n = 197), but showed no significant correlation with tumor size, metastases, nodal status, histological grade, histological type, or estrogen receptor expression. While not achieving statistical significance, there was a trend towards longer survival times in patients with Smad4 negative tumors. CONCLUSION: According to the suggested role of Smad4 as a tumor suppressor we observed that expression of Smad4 is lower in human breast cancer than in surrounding breast epithelium. However, we also observed a trend towards longer survival times in Smad4-negative patients, indicating the complex role of TGF-β signaling in tumor progression

An oestrogen-dependent model of breast cancer created by transformation of normal human mammary epithelial cells

INTRODUCTION: About 70% of breast cancers express oestrogen receptor alpha (ESR1/ERalpha) and are oestrogen-dependent for growth. In contrast with the highly proliferative nature of ERalpha-positive tumour cells, ERalpha-positive cells in normal breast tissue rarely proliferate. Because ERalpha expression is rapidly lost when normal human mammary epithelial cells (HMECs) are grown in vitro, breast cancer models derived from HMECs are ERalpha-negative. Currently only tumour cell lines are available to model ERalpha-positive disease. To create an ERalpha-positive breast cancer model, we have forced normal HMECs derived from reduction mammoplasty tissue to express ERalpha in combination with other relevant breast cancer genes. METHODS: Candidate genes were selected based on breast cancer microarray data and cloned into lentiviral vectors. Primary HMECs prepared from reduction mammoplasty tissue were infected with lentiviral particles. Infected HMECs were characterised by Western blotting, immunofluorescence microscopy, microarray analysis, growth curves, karyotyping and SNP chip analysis. The tumorigenicity of the modified HMECs was tested after orthotopic injection into the inguinal mammary glands of NOD/SCID mice. Cells were marked with a fluorescent protein to allow visualisation in the fat pad. The growth of the graft was analysed by fluorescence microscopy of the mammary glands and pathological analysis of stained tissue sections. Oestrogen dependence of tumour growth was assessed by treatment with the oestrogen antagonist fulvestrant. RESULTS: Microarray analysis of ERalpha-positive tumours reveals that they commonly overexpress the Polycomb-group gene BMI1. Lentiviral transduction with ERalpha, BMI1, TERT and MYC allows primary HMECs to be expanded in vitro in an oestrogen-dependent manner. Orthotopic xenografting of these cells into the mammary glands of NOD/SCID mice results in the formation of ERalpha-positive tumours that metastasise to multiple organs. The cells remain wild type for TP53, diploid and genetically stable. In vivo tumour growth and in vitro proliferation of cells explanted from tumours are dependent on oestrogen. CONCLUSION: We have created a genetically defined model of ERalpha-positive human breast cancer based on normal HMECs that has the potential to model human oestrogen-dependent breast cancer in a mouse and enables the study of mechanisms involved in tumorigenesis and metastasi

Microenvironmental Influences that Drive Progression from Benign Breast Disease to Invasive Breast Cancer

Invasive breast cancer represents the endpoint of a developmental process that originates in the terminal duct lobular units and is believed to progress through stages of increasing proliferation, atypical hyperplasia, and carcinoma in situ before the cancer acquires invasive and metastatic capabilities. By comparison with invasive breast cancer, which has been studied extensively, the preceding stages of benign breast disease are more poorly understood. Much less is known about the molecular changes underlying benign breast disease development and progression, as well as the transition from in situ into invasive disease. Even less focus has been given to the specific role of stroma in this progression. The reasons for lack of knowledge about these lesions often come from their small size and limited sample availability. More challenges are posed by limitations of the models used to investigate the lesions preceding invasive breast cancer. However, recent studies have identified alterations in stromal cell function that may be critical for disease progression from benign disease to invasive cancer: key functions of myoepithelial cells that maintain tissue structure are lost, while tissue fibroblasts become activated to produce proteases that degrade the extracellular matrix and trigger the invasive cellular phenotype. Gene expression profiling of stromal alterations associated with disease progression has also identified key transcriptional changes that occur early in disease development. In this review, we will summarize recent studies showing how stromal factors can facilitate progression of ductal carcinoma in situ to invasive disease. We also suggest approaches to identify processes that control earlier stages of disease progression

Heat and water stress induce unique transcriptional signatures of heat-shock proteins and transcription factors in grapevine

Grapevine is an extremely important crop worldwide. In southern Europe, post-flowering phases of the growth cycle can occur under high temperatures, excessive light, and drought conditions at soil and/or atmospheric level. In this study, we subjected greenhouse grown grapevine, variety Aragonez, to two individual abiotic stresses, water deficit stress (WDS), and heat stress (HS). The adaptation of plants to stress is a complex response triggered by cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Approaches such as array-based transcript profiling allow assessing the expression of thousands of genes in control and stress tissues. Using microarrays, we analyzed the leaf transcriptomic profile of the grapevine plants. Photosynthesis measurements verified that the plants were significantly affected by the stresses applied. Leaf gene expression was obtained using a high-throughput transcriptomic grapevine array, the 23K custom-made Affymetrix Vitis GeneChip. We identified 1,594 genes as differentially expressed between control and treatments and grouped them into ten major functional categories using MapMan software. The transcriptome of Aragonez was more significantly affected by HS when compared with WDS. The number of genes coding for heat-shock proteins and transcription factors expressed solely in response to HS suggesting their expression as unique signatures of HS. However, a cross-talk between the response pathways to both stresses was observed at the level of AP2/ERF transcription factors