A strict regulation of hepatocyte growth factor/scatter factor (HGF/SF)-Met signaling is essential for its appropriate function. Several negative regulators of Met signaling have been identified. Here we report that human Spry2 is induced by HGF/SF and negatively regulates HGF/SF-Met signaling. We show that overexpression of Spry2 inhibits cell proliferation, anchorage-independent cell growth, and migration in wound-healing and in vitro invasion assays. Measured in an electric cell-substrate impedance sensing biosensor, cell movement is restricted, because Spry2 dramatically facilitates cell attachment and spreading by enhancing focal adhesions and increasing stress fibers. An analysis of cell cycle distribution shows, unexpectedly, that Spry2-GFP cells are polyploid. Thus, as with FGF and EGF receptors, Spry2-GFP tempers downstream Met signaling in addition to its pronounced effect on cell adhesion, and it has properties suitable to be considered a tumor-suppressor protein.
Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. METHODS: First, we modified the Tet-pLKO-Puro vector to make it easy ("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods.
How prostate epithelial cells differentiate and how dysregulation of this process contributes to prostate tumorigenesis remain unclear. We recently identified a Myc target and chromatin reader protein, ING4, as a necessary component of human prostate luminal epithelial cell differentiation, which is often lost in primary prostate tumors. Furthermore, loss of ING4 in the context of oncogenic mutations is required for prostate tumorigenesis. Identifying the gene targets of ING4 can provide insight into how its loss disrupts differentiation and leads to prostate cancer.
KAI1/CD82, a tetraspanin protein, was first identified as a metastasis suppressor in prostate cancer. How loss of CD82 expression promotes cancer metastasis is unknown. Restoration of CD82 expression to physiological levels in the metastatic prostate cell line PC3 inhibits integrin-mediated cell migration and invasion, but does not affect integrin expression. Integrin-dependent activation of the receptor kinase c-Met is dramatically reduced in CD82-expressing cells, as is c-Met activation by its ligand HGF/SF. CD82 expression also reduced integrin-induced activation and phosphorylation of the cytoplasmic tyrosine kinase Src, and its downstream substrates p130Cas and FAK Y861. Inhibition of c-Met expression or Src kinase function reduced matrigel invasion of PC3 cells to the same extent as CD82 expression. These data indicate that CD82 functions to suppress integrin-induced invasion by regulating signaling to c-Met and Src kinases, and suggests that CD82 loss may promote metastasis by removing a negative regulator of c-Met and Src signaling.
In vivo in the prostate gland, basal epithelial cells adhere to laminin 5 (LM5) via alpha3beta1 and alpha6beta4 integrins. When placed in culture primary prostate basal epithelial cells secrete and adhere to their own LM5-rich matrix. Adhesion to LM5 is required for cell survival that is dependent on integrin-mediated, ligand-independent activation of the epidermal growth factor receptor (EGFR) and the cytoplasmic tyrosine kinase Src, but not PI-3K. Integrin-mediated adhesion via alpha3beta1, but not alpha6beta4 integrin, supports cell survival through EGFR by signaling downstream to Erk. PC3 cells, which do not activate EGFR or Erk on LM5-rich matrices, are not dependent on this pathway for survival. PC3 cells are dependent on PI-3K for survival and undergo caspase-dependent death when PI-3K is inhibited. The death induced by inhibition of EGFR or Src in normal primary prostate cells is not mediated through or dependent on caspase activation, but depends on the induction of reactive oxygen species. In addition the presence of an autophagic pathway, maintained by adhesion to matrix through alpha3beta1 and alpha6beta4, prevents the induction of caspases when EGFR or Src is inhibited. Suppression of autophagy is sufficient to induce caspase activation and apoptosis in LM5-adherent primary prostate epithelial cells.
