Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.

C-Raf-1 is a major effector of Ras proteins, responsible for activation of the ERK MAP kinase pathway and a critical regulator of both normal growth and oncogenic transformation. Using an inducible form of Raf in MDCK cells, we have shown that sustained activation of Raf alone is able to induce the transition from an epithelial to a mesenchymal phenotype. Raf promoted invasive growth in collagen gels, a characteristic of malignant cells; this was dependent on the operation of an autocrine loop involving TGFβ, whose secretion was induced by Raf. TGFβ induced growth inhibition and apoptosis in normal MDCK cells: Activation of Raf led to inhibition of the ability of TGFβ to induce apoptosis but not growth retardation. ERK has been reported previously to inhibit TGFβ signaling via phosphorylation of the linker region of Smads, which prevents their translocation to the nucleus. However, we found no evidence in this system that ERK can significantly influence the function of Smad2, Smad3, and Smad4 at the level of nuclear translocation, DNA binding, or transcriptional activation. Instead, strong activation of Raf caused a broad protection of these cells from various apoptotic stimuli, allowing them to respond to TGFβ with increased invasiveness while avoiding cell death. The Raf–MAP kinase pathway thus synergizes with TGFβ in promoting malignancy but does not directly impair TGFβ-induced Smad signaling.

The role of phagocytosis in the neuroprotective function of microglia has been appreciated for a long time, but only more recently a dysregulation of this process has been recognized in Parkinson’s disease (PD). Indeed, microglia play several critical roles in central nervous system (CNS), such as clearance of dying neurons and pathogens as well as immunomodulation, and to fulfill these complex tasks they engage distinct phenotypes. Regulation of phenotypic plasticity and phagocytosis in microglia can be impaired by defects in molecular machinery regulating critical homeostatic mechanisms, including autophagy. Here, we briefly summarize current knowledge on molecular mechanisms of microglia phagocytosis, and the neuro-pathological role of microglia in PD. Then we focus more in detail on the possible functional role of microglial phagocytosis in the pathogenesis and progression of PD. Evidence in support of either a beneficial or deleterious role of phagocytosis in dopaminergic degeneration is reported. Altered expression of target-recognizing receptors and lysosomal receptor CD68, as well as the emerging determinant role of α-synuclein (α-SYN) in phagocytic function is discussed. We finally discuss the rationale to consider phagocytic processes as a therapeutic target to prevent or slow down dopaminergic degeneration.

Oncogenic Ras interferes with adhesive functions of epithelial cells, but requires tumor growth factor β (TGFβ) signaling to cause epithelial–mesenchymal transition (EMT) and tumor progression in model systems. To investigate the mechanisms by which Ras and TGFβ pathways cooperate in EMT induction, we introduced a tamoxifen-inducible version of Raf-1 (RafER) into fully polarized, mammary epithelial cells (EpH4). EMT characterized by loss of E-cadherin expression and upregulation of invasiveness-promoting genes was induced by TGFβ plus 4-hydroxytamoxifen (4HT) activation of RafER. Downregulation of E-cadherin by RafER plus TGFβ was detectable in total cell lysates after 48 h and much earlier in detergent-insoluble fractions of E-cadherin. Both pathways cooperated to strongly enhance endocytosis of E-cadherin, mainly via the clathrin-dependent route. Pulse-chase experiments showed decreased E-cadherin protein stability in cells stimulated with TGFβ and 4HT and increased E-cadherin half-life in the presence of monensin. Monensin and chloroquine prevented E-cadherin degradation to different extent, but only monensin effectively blocked the loss of E-cadherin from the junctional complexes. Both lysosome inhibitors caused accumulation of E-cadherin vesicles, some of which were positive for Cathepsin D and lysosome-associated membrane protein 1 (LAMP-1). In addition, TGFβ and mitogen-activated protein kinase hyperactivation synergistically induced E-cadherin ubiquitination, suggesting that the cooperation of Raf and TGFβ favors lysosomal degradation of E-cadherin instead of its recycling. Our data indicate that early stages of EMT involve cooperative, post-translational downregulation of E-cadherin, whereas loss of E-cadherin via transcriptional repression is a late event in EMT.