Autocrine CSF-1R activation promotes Src-dependent disruption of mammary epithelial architecture.

Elevated coexpression of colony-stimulating factor receptor (CSF-1R) and its ligand, CSF-1, correlates with invasiveness and poor prognosis of a variety of epithelial tumors (Kacinski, B.M. 1995. Ann. Med. 27:79-85). Apart from recruitment of macrophages to the tumor site, the mechanisms by which CSF-1 may potentiate invasion are poorly understood. We show that autocrine CSF-1R activation induces hyperproliferation and a profound, progressive disruption of junctional integrity in acinar structures formed by human mammary epithelial cells in three-dimensional culture. Acini coexpressing receptor and ligand exhibit a dramatic relocalization of E-cadherin from the plasma membrane to punctate intracellular vesicles, accompanied by its loss from the Triton-insoluble fraction. Interfering with Src kinase activity, either by pharmacological inhibition or mutation of the Y561 docking site on CSF-1R, prevents E-cadherin translocation, suggesting that CSF-1R disrupts cell adhesion by uncoupling adherens junction complexes from the cytoskeleton and promoting cadherin internalization through a Src-dependent mechanism. These findings provide a mechanistic basis whereby CSF-1R could contribute to invasive progression in epithelial cancers.

Novel role for PDEF in epithelial cell migration and invasion.

Cell migration and invasion are two critical cellular processes that are often deregulated during tumorigenesis. To identify factors that contribute to oncogenic progression by stimulating cell migration, we conducted a powerful retroviral based migration screen using an MCF7 cDNA library and the immortalized human breast epithelial cell line MCF-10A. We identified prostate derived Ets factor (PDEF), an Ets transcription factor that is overexpressed in both prostate and breast carcinoma, as a candidate promigratory gene from this screen. Whereas PDEF induced limited motility of MCF-10A cells, coexpression of PDEF with the receptor tyrosine kinases (RTK) ErbB2 and colony-stimulating factor receptor (CSF-1R)/CSF-1 significantly enhanced MCF-10A motility. Furthermore, cells coexpressing PDEF with either ErbB2 or CSF-1R/CSF-1 induced a dramatic invasive phenotype in three-dimensional cultures. Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway also enhanced PDEF-induced motility and invasion, suggesting that activation of the ERK/mitogen-activated protein kinase by ErbB2 and CSF-1R/CSF-1 can cooperate with PDEF to promote motility and invasion. Furthermore, PDEF promoted anchorage-independent growth of ErbB2 and CSF-1R/CSF-1-expressing cells. Using laser capture microdissection, we also found that PDEF mRNA is overexpressed in breast tumor epithelia throughout tumor progression. Taken together, these findings suggest that the transcription factor PDEF may play an important role in breast tumorigenesis and that PDEF overexpression may be particularly significant in tumors that exhibit activation of oncogenic RTKs such as ErbB2 and CSF-1R.

Profiling Y561-dependent and -independent substrates of CSF-1R in epithelial cells.

Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.

Persistent expression of stabilized beta-catenin delays maturation of radial glial cells into intermediate progenitors.

Transgenic mice expressing stabilized beta-catenin in neural progenitors develop enlarged brains resulting from increased progenitor expansion. To more precisely define beta-catenin regulation of progenitor fate, we employed a conditional transgenic approach to delete the beta-catenin regulatory domain from neural progenitors, resulting in expression of stabilized protein from its endogenous promoter in these cells and their progeny. An increased fraction of transgenic cortical cells express the progenitor markers Nestin and LewisX, confirming a relative expansion of this population. Sustained beta-catenin activity expands RC2 and Pax6 expression in the developing cortex while postponing the onset of Tbr2 expression, suggesting a delay in maturation of radial glia into intermediate progenitors. Furthermore, transgenic cortical cells fail to either upregulate ErbB4 or develop a mitogenic response to epidermal growth factor, changes that normally accompany the acquisition of an intermediate fate. Likewise, transgenic brains do not develop a distinct subventricular zone or superficial cortical layers, and overexpression of stabilized beta-catenin by in utero electroporation caused a relative reduction of upper layer vs. lower layer cortical neurons, indicating that persistent beta-catenin activity interferes with the generation of progenitors responsible for the production of upper layer cortical neurons. Collectively, these findings demonstrate that beta-catenin functions to maintain the radial glial population, and suggest that downregulation of beta-catenin signaling may be critical to facilitate the transition to an intermediate progenitor phenotype.

Use of three-dimensional basement membrane cultures to model oncogene-induced changes in mammary epithelial morphogenesis.

The development of breast carcinomas involves a complex set of phenotypic alterations in breast epithelial cells and the surrounding microenvironment. While traditional transformation assays provide models for investigating certain aspects of the cellular processes associated with tumor initiation and progression, they do not model alterations in tissue architecture that are critically involved in tumor development. In this review, we provide examples of how three-dimensional (3D) cell culture models can be utilized to dissect the pathways involved in the development of mammary epithelial structures and to elucidate the mechanisms responsible for oncogene-induced phenotypic alterations in epithelial behavior and architecture. Many normal mammary epithelial cell lines undergo a stereotypic morphogenetic process when grown in the presence of exogenous matrix proteins. This 3D morphogenesis culminates in the formation of well-organized, polarized spheroids, and/or tubules that are highly reminiscent of normal glandular architecture. In contrast, transformed cell lines isolated from mammary tumors exhibit significant deviations from normal epithelial behavior in 3D culture. We describe the use of 3D models as a method for both reconstructing and deconstructing the cell biological and biochemical events involved in mammary neoplasia.