The origins of the mesenchymal cells participating in tissue repair and pathological processes, notably tissue fibrosis, tumor invasiveness, and metastasis, are poorly understood. However, emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) represent one important source of these cells. As we discuss here, processes similar to the EMTs associated with embryo implantation, embryogenesis, and organ development are appropriated and subverted by chronically inflamed tissues and neoplasias. The identification of the signaling pathways that lead to activation of EMT programs during these disease processes is providing new insights into the plasticity of cellular phenotypes and possible therapeutic interventions.

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The basics of epithelial-mesenchymal transition

Without epithelial–mesenchymal transitions, in which polarized epithelial cells are converted into motile cells, multicellular organisms would be incapable of getting past the blastula stage of embryonic development. However, this important developmental programme has a more sinister role in tumour progression. Epithelial–mesenchymal transition provides a new basis for understanding the progression of carcinoma towards dedifferentiated and more malignant states.

Epithelial–mesenchymal transitions in tumour progression

The heparan sulfate on the surface of all adherent cells modulates the actions of a large number of extracellular ligands. Members of both cell surface heparan sulfate proteoglycan families, the transmembrane syndecans and the glycosylphosphoinositide-linked glypicans, bind these ligands and enhance formation of their receptor-signaling complexes. These heparan sulfate proteoglycans also immobilize and regulate the turnover of ligands that act at the cell surface. The extracellular domains of these proteoglycans can be shed from the cell surface, generating soluble heparan sulfate proteoglycans that can inhibit interactions at the cell surface. Recent analyses of genetic defects in Drosophila melanogaster, mice, and humans confirm most of these activities in vivo and identify additional processes that involve cell surface heparan sulfate proteoglycans. This chapter focuses on the mechanisms underlying these activities and on the cellular functions that they regulate.

Functions of Cell Surface Heparan Sulfate Proteoglycans

MicroRNAs have been implicated in regulating diverse cellular pathways. Although there is emerging evidence that some microRNAs can function as oncogenes or tumour suppressors, the role of microRNAs in mediating cancer metastasis remains unexplored. Here we show, using a combination of mouse and human cells, that microRNA-10b (miR-10b) is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion. Overexpression of miR-10b in otherwise non-metastatic breast tumours initiates robust invasion and metastasis. Expression of miR-10b is induced by the transcription factor Twist, which binds directly to the putative promoter of mir-10b (MIRN10B). The miR-10b induced by Twist proceeds to inhibit translation of the messenger RNA encoding homeobox D10, resulting in increased expression of a well-characterized pro-metastatic gene, RHOC. Significantly, the level of miR-10b expression in primary breast carcinomas correlates with clinical progression. These findings suggest the workings of an undescribed regulatory pathway, in which a pleiotropic transcription factor induces expression of a specific microRNA, which suppresses its direct target and in turn activates another pro-metastatic gene, leading to tumour cell invasion and metastasis.

Tumour invasion and metastasis initiated by microRNA-10b in breast cancer

Epithelial and hematopoietic cells have a high turnover and their progenitor cells divide continuously, making them prime targets for genetic and epigenetic changes that lead to cell transformation and tumorigenesis. The consequent changes in cell behavior and responsiveness result not only from genetic alterations such as activation of oncogenes or inactivation of tumor suppressor genes, but also from altered production of, or responsiveness to, stimulatory or inhibitory growth and differentiation factors. Among these, transforming growth factor β (TGF-β) and its signaling effectors act as key determinants of carcinoma cell behavior. The autocrine and paracrine effects of TGF-β on tumor cells and the tumor micro-environment exert both positive and negative influences on cancer development. Accordingly, the TGF-β signaling pathway has been considered as both a tumor suppressor pathway and a promoter of tumor progression and invasion. Here we evaluate the role of TGF-β in tumor development and attempt to reconcile the positive and negative effects of TGF-β in carcinogenesis.

TGF-beta signaling in tumor suppression and cancer progression.

Metastasis of epithelial tumor cells can be associated with the acquisition of fibroblastoid features and the ability to invade stroma and blood vessels. Using matched in vivo and in vitro culture systems employing fully polarized, mammary epithelial cells, we report here that TGF-beta1 brings about these changes in Ras-transformed cells but not in normal cells. When grown in collagen gels in the absence of TGF-beta, both normal and Ras-transformed mammary epithelial cells form organ-like structures in which the cells maintain their epithelial characteristics. Under these conditions, treatment of normal cells with TGF-beta results in growth arrest. The same treatment renders Ras-transformed epithelial cells fibroblastoid, invasive, and resistant to growth inhibition by TGF-beta. After this epithelial-fibroblastoid conversion, the Ras-transformed cells start to secrete TGF-beta themselves, leading to autocrine maintenance of the invasive phenotype and recruitment of additional cells to become fibroblastoid and invasive. More important, this cooperation of activated Ha-Ras with TGF-beta1 is operative during in vivo tumorigenesis and, as in wound healing processes, is dependent on epithelial-stromal interactions.

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TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells.

Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion.

Members of the epidermal growth factor (EGF) receptor family are known to be specifically involved in mammary carcinogenesis. As a nuclear target of activated receptors, we examined c-Jun in mammary epithelial cells. For this, we used a c-JunER fusion protein which was tightly controlled by estrogen. Activation of the JunER by hormone resulted in the transcriptional regulation of a variety of AP-1 target genes. Hormone-activated JunER induced the loss of epithelial polarity, a disruption of intercellular junctions and normal barrier function and the formation of irregular multilayers. These changes were completely reversible upon hormone withdrawal. Loss of epithelial polarity involved redistribution of both apical and basolateral proteins to the entire plasma membrane. The redistribution of E-cadherin and beta-catenin was accompanied by a destabilization of complexes formed between these two proteins, leading to an enrichment of beta-catenin in the detergent-soluble fraction. Uninduced cells were able to form three-dimensional tubular structures in collagen I gels which were disrupted upon JunER activation, leading to irregular cell aggregates. The JunER-induced disruption of tubular structures was dependent on active signaling by growth factors. Moreover, the effects of JunER could be mimicked in normal cells by the addition of acidic fibroblast growth factor (aFGF). These data suggest that a possible function of c-Jun in epithelial cells is to modulate epithelial polarity and regulate tissue organization, processes which may be equally important for both normal breast development and as initiating steps in carcinogenesis.

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The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions.

Pharmaceutical compositions containing as active compound a substance which inhibits the activity of TGFβ on tumour cells of epithelial origin, for the treatment of epithelial, invasive tumour diseases which are characterized by a reversible transition of the cells from an epithelial, non-invasive state into a fibroblastoid, invasive state The pharmaceutical composition contains a TGFβ inhibitor, preferably combined with an Ras inhibitor Process for screening substances for the treatment of epithelial, invasive tumour diseases

Ernst Reichmann

Papers

TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells.

Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion.

The estrogen-dependent c-JunER protein causes a reversible loss of mammary epithelial cell polarity involving a destabilization of adherens junctions.

Methods of screening for pharmacologically active compounds for the treatment of tumour diseases

Three-Dimensional Organotypic Growth of Epithelial Cells in Reconstituted Extracellular Matrix.