The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Mesenchymal stem cells have been recently described to localize to breast carcinomas, where they integrate into the tumour-associated stroma. However, the involvement of mesenchymal stem cells (or their derivatives) in tumour pathophysiology has not been addressed. Here, we demonstrate that bone-marrow-derived human mesenchymal stem cells, when mixed with otherwise weakly metastatic human breast carcinoma cells, cause the cancer cells to increase their metastatic potency greatly when this cell mixture is introduced into a subcutaneous site and allowed to form a tumour xenograft. The breast cancer cells stimulate de novo secretion of the chemokine CCL5 (also called RANTES) from mesenchymal stem cells, which then acts in a paracrine fashion on the cancer cells to enhance their motility, invasion and metastasis. This enhanced metastatic ability is reversible and is dependent on CCL5 signalling through the chemokine receptor CCR5. Collectively, these data demonstrate that the tumour microenvironment facilitates metastatic spread by eliciting reversible changes in the phenotype of cancer cells.

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During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.

Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into nonstem progeny in a unidirectional manner. Here we identify a subpopulation of basal-like human mammary epithelial cells that departs from that assumption, spontaneously dedifferentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that nonstem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of posttransformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bidirectional interconversions between stem and nonstem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer.breast cancer | dedifferentiation

The proteins Grb2-Sem-5, Shc and Sos have been implicated in the signalling pathway from tyrosine kinase receptors to Ras. Grb2-Sem-5 binds directly to murine Sos1, a Ras exchange factor, through two SH3 domains. Sos is also associated with ligand-activated tyrosine kinase receptors which bind Grb2-Sem-5, and with the Grb2-Sem-5 binding protein, Shc. Ectopic expression of Drosophila Sos stimulates morphological transformation of rodent fibroblasts. These data define a pathway by which tyrosine kinases act through Ras to control cell growth and differentiation.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.