Keith R. Johnson

Cadherin switching: essential for behavioral but not morphological changes during an epithelium-to- mesenchyme transition

Epithelium-to-mesenchyme transitions (EMTs) are characterized by morphological and behavioral changes in cells. During an EMT, E-cadherin is downregulated while N-cadherin is upregulated. The goal of this study was to understand the role cadherin switching plays in EMT using a classical model system: transforming growth factor β1 (TGF-β1)-mediated EMT in mammary epithelial cells. We showed that stress fibers and focal adhesions are increased, and cell-cell junctions are decreased in response to TGF-β1. Moreover, these changes were reversible upon removal of TGF-β1. Downregulation of E-cadherin and upregulation of N-cadherin were both transcriptional. Neither experimental knockdown nor experimental overexpression of N-cadherin interfered with the morphological changes. In addition, the morphological changes associated with EMT preceded the downregulation of E-cadherin. Interestingly, TGF-β1-induced motility in N-cadherin-knockdown cells was significantly reduced. Together, these data suggest that cadherin switching is necessary for increased motility but is not required for the morphological changes that accompany EMT.

Cadherin-mediated cellular signaling

Recent cadherin studies focusing on cellular signaling have shown that several pathways are activated by cadherin-mediated cell-cell contact. Cadherin-mediated contacts activate Rho family GTPases, regulate the availability of beta-catenin to participate in Wnt signaling, and function in receptor tyrosine kinase signaling. Although different classical cadherins bind to the same cytosolic proteins via their cytoplasmic tails, one message that is clear from the recent literature is that downstream signals emanating from cadherin-mediated contacts are both cadherin-specific and cell-context-specific.

Nuclear association of the cytoplasmic tail of MUC1 and β-catenin

MUC1, an integral membrane mucin associated with the metastatic phenotype, is overexpressed by most human carcinoma cells. The MUC1 cytoplasmic tail (CT) is postulated to function in morphogenetic signal transduction via interactions with Grb2/Sos, c-Src, and β-catenin. We investigated intracellular trafficking of the MUC1 CT, using epitope-tagged constructs that were overexpressed in human pancreatic cancer cell lines S2-013 and Panc-1. The MUC1 CT was detected at the inner cell surface, in the cytosol, and in the nucleus of cells overexpressing MUC1. Fragments of the MUC1 CT were associated with β-catenin in both cytoplasm and nuclei. Overexpression of MUC1 increased steady state levels of nuclear β-catenin but decreased nuclear levels of plakoglobin (γ-catenin). There was no detectable association between plakoglobin and the MUC1 CT. Coimmunoprecipitation experiments revealed that the cytoplasmic and nuclear association of MUC1 CT and β-catenin was not affected by disruption of Ca2+-dependent intercellular cadherin interactions. These results demonstrate nuclear localization of fragments of MUC1 CT in association with β-catenin and raise the possibility that overexpression of the MUC1 CT stabilizes β-catenin and enhances levels of nuclear β-catenin during disruption of cadherin-mediated cell-cell adhesion.

Collagen I–mediated up-regulation of N-cadherin requires cooperative signals from integrins and discoidin domain receptor 1

Tumor cells undergo epithelial-to-mesenchymal transition (EMT) to convert from a benign to a malignant phenotype. Our recent focus has been signaling pathways that promote EMT in response to collagen. We have shown that human pancreatic cancer cells respond to collagen by up-regulating N-cadherin, which promotes tumor growth, invasion, and metastasis. Initial characterization showed that knocking down c-Jun NH2-terminal kinase prevented N-cadherin up-regulation and limited tumor growth and invasion in a mouse model for pancreatic cancer. The current study was designed to understand the pathway from collagen to N-cadherin up-regulation. Initiation of the signal requires two collagen receptors, α2β1 integrin and discoidin domain receptor (DDR) 1. Each receptor propagates signals through separate pathways that converge to up-regulate N-cadherin. Focal adhesion kinase (FAK)–related protein tyrosine kinase (Pyk2) is downstream of DDR1, whereas FAK is downstream of α2β1 integrin. Both receptor complexes rely on the p130 Crk-associated substrate scaffold. Interestingly, Rap1, but not Rho family guanosine triphosphatases, is required for the response to collagen I.

Collagen I Promotes Metastasis in Pancreatic Cancer by Activating c-Jun NH2-Terminal Kinase 1 and Up-regulating N-Cadherin Expression

We have previously shown that N-cadherin expression is associated with tumor invasion, and that some cancer cells respond to specific extracellular matrix molecules by up-regulating N-cadherin. Pancreatic cancer is characterized by excessive deposition of type I collagen. Here, we show that human pancreatic cancer cells respond to collagen I, but not other matrices, by increasing motility and up-regulating mesenchymal markers, including N-cadherin. Both collagen I-mediated motility and metastasis in a mouse model for pancreatic cancer were inhibited by N-cadherin knockdown. Furthermore, inhibiting c-Jun NH(2)-terminal kinase (JNK) with chemical inhibitors or short hairpin RNA abrogated all collagen I-induced changes. We show that JNK1 is activated in response to collagen I, which increases tumorigenesis by up-regulating N-cadherin expression and by increasing motility.

MUC1 mucin stabilizes and activates hypoxia-inducible factor 1 alpha to regulate metabolism in pancreatic cancer

Aberrant glucose metabolism is one of the hallmarks of cancer that facilitates cancer cell survival and proliferation. Here, we demonstrate that MUC1, a large, type I transmembrane protein that is overexpressed in several carcinomas including pancreatic adenocarcinoma, modulates cancer cell metabolism to facilitate growth properties of cancer cells. MUC1 occupies the promoter elements of multiple genes directly involved in glucose metabolism and regulates their expression. Furthermore, MUC1 expression enhances glycolytic activity in pancreatic cancer cells. We also demonstrate that MUC1 expression enhances in vivo glucose uptake and expression of genes involved in glucose uptake and metabolism in orthotopic implantation models of pancreatic cancer. The MUC1 cytoplasmic tail is known to activate multiple signaling pathways through its interactions with several transcription factors/coregulators at the promoter elements of various genes. Our results indicate that MUC1 acts as a modulator of the hypoxic response in pancreatic cancer cells by regulating the expression/stability and activity of hypoxia-inducible factor-1α (HIF-1α). MUC1 physically interacts with HIF-1α and p300 and stabilizes the former at the protein level. By using a ChIP assay, we demonstrate that MUC1 facilitates recruitment of HIF-1α and p300 on glycolytic gene promoters in a hypoxia-dependent manner. Also, by metabolomic studies, we demonstrate that MUC1 regulates multiple metabolite intermediates in the glucose and amino acid metabolic pathways. Thus, our studies indicate that MUC1 acts as a master regulator of the metabolic program and facilitates metabolic alterations in the hypoxic environments that help tumor cells survive and proliferate under such conditions.

Impaired Trafficking of Connexins in Androgen-independent Human Prostate Cancer Cell Lines and Its Mitigation by α-Catenin

Gap junctions, composed of connexins, provide a pathway of direct intercellular communication for the diffusion of small molecules between cells. Evidence suggests that connexins act as tumor suppressors. We showed previously that expression of connexin-43 and connexin-32 in an indolent prostate cancer cell line, LNCaP, resulted in gap junction formation and growth inhibition. To elucidate the role of connexins in the progression of prostate cancer from a hormone-dependent to -independent state, we introduced connexin-43 and connexin-32 into an invasive, androgen-independent cell line, PC-3. Expression of these proteins in PC-3 cells resulted in intracellular accumulation. Western blot analysis revealed a lack of Triton-insoluble, plaque-assembled connexins. In contrast to LNCaP cells, connexins could not be cell surface-biotinylated and did not reside in the cell surface derived endocytic vesicles, in PC-3 cells, suggesting impaired trafficking to the cell surface. Intracellular accumulation of connexins was observed in several androgen-independent prostate cancer cell lines. Transient expression of α-catenin facilitated the trafficking of both connexins to the cell surface and induced gap junction assembly. Our results suggest that impaired trafficking, and not the inability to form gap junctions, is the major cause of communication deficiency in human prostate cancer cell lines.

Expression of inappropriate cadherins by epithelial tumor cells promotes endocytosis and degradation of E-cadherin via competition for p120ctn

Cadherin cell–cell adhesion proteins play an important role in modulating the behavior of tumor cells. E-cadherin serves as a suppressor of tumor cell invasion, and when tumor cells turn on the expression of a non-epithelial cadherin, they often express less E-cadherin, enhancing the tumorigenic phenotype of the cells. Here, we show that when A431 cells are forced to express R-cadherin, they dramatically downregulate the expression of endogenous E- and P-cadherin. In addition, we show that this downregulation is owing to increased turnover of the endogenous cadherins via clathrin-dependent endocytosis. p120ctn binds to the juxtamembrane domain of classical cadherins and has been proposed to regulate cadherin adhesive activity. One way p120ctn may accomplish this is to serve as a rheostat to regulate the levels of cadherin. Here, we show that the degradation of E-cadherin in response to expression of R-cadherin is owing to competition for p120ctn.

ADH‐1 suppresses N‐cadherin‐dependent pancreatic cancer progression

Pancreatic cancer is one of the most aggressive malignant diseases. We recently reported that N‐cadherin plays a key role in tumor progression and metastasis in pancreatic cancer. For this study, we sought to determine if an N‐cadherin‐blocking peptide (ADH‐1) could prevent N‐cadherin‐mediated tumor progression in a mouse model for pancreatic cancer. The effect of ADH‐1 on N‐cadherin‐mediated cell scattering and migration on collagen I was examined using pancreatic cancer cells. We also examined the influence of ADH‐1 on cell apoptosis. Furthermore, in vivo animal studies were performed using orthotopic injection of N‐cadherin overexpressing BxPC‐3 cells with or without ADH‐1 treatment. BxPC‐3 and Capan‐1 cells exhibited increased expression of N‐cadherin in response to collagen I. This increase in N‐cadherin promoted cell scattering and migration in response to collagen I. ADH‐1 prevented these changes, but did not inhibit upregulation of N‐cadherin. TUNEL assays and immunoblots for caspase‐3 showed that ADH‐1 induced apoptosis in a concentration dependent and N‐cadherin dependent manner in pancreatic cancer cells. ADH‐1 treatment resulted in significant reductions in tumor growth and lung metastasis in a mouse model for pancreatic cancer. The N‐cadherin antagonist, ADH‐1 has significant antitumor activity against N‐cadherin‐expressing cells using in vitro assays and in an orthotopic mouse model for pancreatic cancer, raising the possibility that N‐cadherin antagonists have therapeutic potential for the treatment of pancreatic cancer in humans.

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

In tumor cells that coexpress Cx43 and Cx26, the assembly of Cx43 is selectively impaired due to endocytosis. Assembly can be restored upon expressing a Cx43 sorting-motif mutant and mutants that cannot be phosphorylated on Ser-279 or Ser-282.