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.

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. p120 ctn binds to the juxtamembrane domain of classical cadherins and has been proposed to regulate cadherin adhesive activity. One way p120 ctn 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 p120 ctn .

Desmosomes are prominent adhesive junctions found in various epithelial tissues. The cytoplasmic domains of desmosomal cadherins interact with a host of desmosomal plaque proteins, including plakophilins, plakoglobin and desmoplakin, which, in turn, recruit the intermediate filament cytoskeleton to sites of cell-cell contact. Although the individual components of the desmosome are known, mechanisms regulating the assembly of this junction are poorly understood. Protein palmitoylation is a posttranslational lipid modification that plays an important role in protein trafficking and function. Here, we demonstrate that multiple desmosomal components are palmitoylated in vivo. Pharmacologic inhibition of palmitoylation disrupts desmosome assembly at cell-cell borders. We mapped the site of plakophilin palmitoylation to a conserved cysteine residue present in the armadillo repeat domain. Mutation of this single cysteine residue prevents palmitoylation, disrupts plakophilin incorporation into the desmosomal plaque and prevents plakophilindependent desmosome assembly. Finally, plakophilin mutants unable to become palmitoylated act in a dominant-negative manner to disrupt proper localization of endogenous desmosome components and decrease desmosomal adhesion. Taken together, these data demonstrate that palmitoylation of desmosomal components is important for desmosome assembly and adhesion.

Desmosomes are prominent adhesive junctions present between many epithelial cells as well as cardiomyocytes. The mechanisms controlling desmosome assembly and remodeling in epithelial and cardiac tissue are poorly understood. We recently identified protein palmitoylation as a mechanism regulating desmosome dynamics. In this study, we have focused on the palmitoylation of the desmosomal cadherin desmoglein-2 (Dsg2) and characterized the role that palmitoylation of Dsg2 plays in its localization and stability in cultured cells. We identified two cysteine residues in the juxtamembrane (intracellular anchor) domain of Dsg2 that, when mutated, eliminate its palmitoylation. These cysteine residues are conserved in all four desmoglein family members. Although mutant Dsg2 localizes to endogenous desmosomes, there is a significant delay in its incorporation into junctions, and the mutant is also present in a cytoplasmic pool. Triton X-100 solubility assays demonstrate that mutant Dsg2 is more soluble than wild-type protein. Interestingly, trafficking of the mutant Dsg2 to the cell surface was delayed, and a pool of the non-palmitoylated Dsg2 co-localized with lysosomal markers. Taken together, these data suggest that palmitoylation of Dsg2 regulates protein transport to the plasma membrane. Modulation of the palmitoylation status of desmosomal cadherins can affect desmosome dynamics.

Pancreatic ductal adenocarcinomas are highly malignant cancers characterized by extensive invasion into surrounding tissues, metastasis to distant organs, and a limited response to therapy. A main feature of pancreatic ductal adenocarcinomas is desmoplasia, which leads to extensive deposition of collagen I. We have demonstrated that collagen I can induce epithelialmesenchymal transition (EMT) in pancreatic cancer cells. A hallmark of EMT is an increase in the expression of the mesenchymal cadherin N-cadherin. Previously we showed up-regulation of N-cadherin promotes tumor cell invasion and that collagen I-induced EMT is mediated by two collagen receptors, ␣2␤1-integrin and discoidin domain receptor 1 (DDR1). DDR1 is a receptor-tyrosine kinase widely expressed during embryonic development and in many adult tissues and is also highly expressed in many different cancers. In the signaling pathway initiated by collagen, we have shown proline-rich tyrosine kinase 2 (Pyk2) is downstream of DDR1. In this study we found isoform b of DDR1 is responsible for collagen I-induced up-regulation of N-cadherin and tyrosine 513 of DDR1b is necessary. Knocking down Shc1, which binds to tyrosine 513 of DDR1b via its PTB (phosphotyrosine binding) domain, eliminates the upregulation of N-cadherin. The signaling does not require a functional SH2 domain or the tyrosine residues commonly phosphorylated in Shc1 but is mediated by the interaction between a short segment of the central domain of Shc1 and the proline-rich region of Pyk2. Taken together, these data illustrate DDR1b, but not DDR1a, mediates collagen I-induced N-cadherin up-regulation, and Shc1 is involved in this process by coupling to both DDR1 and Pyk2.Pancreatic cancer is currently the fourth leading cause of cancer-related mortality in the United States with a 5-year survival rate of ϳ6% (American Cancer Society, 2014). This unfortunate outcome is mainly due to the aggressive behavior of this cancer and the lack of symptoms early in the disease. One of the major features of pancreatic cancer is that during the early stages an extensive stromal reaction occurs called desmoplasia. A dense stroma forms, which consists of myofibroblasts (pancreatic stellate cells), abundant extracellular matrix, and different types of inflammatory cells including macrophages, mast cells, lymphocytes, and plasma cells (1, 2). The extracellular matrix components include collagens, fibronectin, hyaluronic acid, and proteoglycans, whereas the major part is type I collagen (3, 4).The atypical stroma plays many roles in pancreatic cancer. It changes the normal architecture of pancreatic tissue and induces an abnormal configuration of blood and lymphatic vessels resulting in a hypovascular and hypoxic microenvironment. It also presents a barrier to drug delivery and promotes tumor immunologic tolerance (2, 4). Previously, our laboratory showed that collagen I can induce epithelial-mesenchymal transition (EMT) 2 -like changes in pancreatic cancer mediated by two collagen receptors, ␣2␤1-integrin and ...

Adherens junctions and desmosomes are critical for embryogenesis and the integrity of adult tissues. To form these junctions, classical cadherins interact via α- and β-catenin with the actin cytoskeleton, whereas desmosomal cadherins interact with the intermediate filament system. Here, we used a hormone-activated mutant N-cadherin expressed in fibroblasts to show the existence of a novel classical cadherin adhesion system. N-cadherin was fused at its C-terminus to a modified estrogen receptor ligand-binding domain (NcadER) that binds 4-hydroxytamoxifen (4OHT) and expressed in L cells, which lack an endogenous cadherin. Cells with the mutant cadherin (LNER cells) aggregated in the absence of 4OHT, but only in its presence formed tightly compacted aggregates like those formed by L cells expressing wild-type N-cadherin (LN cells). Compaction of LNER cells treated with 4OHT was accompanied by elevated levels of p120ctn in NcadER immunoprecipitates, compared to immunoprecipitates of non-treated cells, but without changes in α- and β-catenin, or actin. Compaction induced by 4OHT was also accompanied by increased interaction of the NcadER with the cytoskeleton and increased vimentin organization. Vimentin co-immunoprecipitated with the NcadER/catenin complex, suggesting an interaction between cadherin and vimentin. The mechanism by which vimentin interacts with the cadherin appears to involve p120ctn as it co-immunoprecipitates and colocalizes with vimentin in the parent L cells, which lack a cadherin and α- and β-catenins. Disrupting the actin cytoskeleton with cytochalasin B inhibited aggregation, whereas knocking down vimentin with specific siRNAs inhibited compaction. Based on our results we propose that a vimentin-based classical cadherin complex functions together with the actin-based complex to promote strong cell-cell adhesion in fibroblasts.

KRAS is the most commonly mutated oncogene; targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells within the heterogeneous tumor population and/or acquired mutations that reactivate RTK/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 or KSR1 both enhances the efficacy of and prevents resistance to the MEK inhibitor trametinib in KRAS-mutated lung and colorectal adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting TICs, but only in KRASG12- or KRASG13-mutated LUAD and COAD cell lines that lacked PIK3CA co-mutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to combination therapy with trametinib and BI-3406. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented treatment-induced TIC upregulation and restored trametinib sensitivity across KRAS mutant cell lines in both PIK3CA-mutated and PIK3CA wildtype cancers. Our findings demonstrate that vertical targeting of RTK/RAS signaling is an effective strategy to target KRAS-mutated cancers, but the specific combination is dependent both on the specific KRAS mutant and underlying co-mutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations.

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here we show that KSR1 and ERK promote EMT-like phenotype through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E- to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT-like phenotype, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E- to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT-like phenotype via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.