ABSTRACT
In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells.
Subject(s)
Cadherins/metabolism , Carcinoma/metabolism , Epithelial Cells/metabolism , Epithelial-Mesenchymal Transition , ErbB Receptors/metabolism , Animals , Cadherins/genetics , Carcinoma/genetics , Carcinoma/pathology , Dogs , Epithelial Cells/pathology , ErbB Receptors/genetics , Madin Darby Canine Kidney Cells , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Toward the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of tert-butyl methacrylate (tBMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of tert-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of â¼95 µm by day 28. Cells in these structures were viable, formed cell-cell contacts through E-cadherin (E-CAD), and displayed cortical organization of F-actin. Compared with the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids, and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD are a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs.
Subject(s)
Carcinogenesis/drug effects , Hydrogels/chemistry , Peptides/chemistry , Polymers/chemistry , Biomimetics , Cell Adhesion/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Humans , Hyaluronic Acid/chemistry , Hyaluronic Acid/pharmacology , Hydrogels/chemical synthesis , Hydrogels/pharmacology , Male , Methacrylates/chemical synthesis , Methacrylates/chemistry , Methacrylates/pharmacology , Peptides/chemical synthesis , Peptides/pharmacology , Polymers/chemical synthesis , Polymers/pharmacology , Prostatic Neoplasms/chemically induced , Prostatic Neoplasms/pathologyABSTRACT
Nanomedicine has advanced to clinical trials for adult cancer therapy. However, the field is still in its infancy for treatment of childhood malignancies such as acute lymphoblastic leukemia (ALL). Nanotherapy offers multiple advantages over conventional therapy. It facilitates targeted delivery and enables controlled release of drugs to reduce treatment-related side effects. Here, we demonstrate that doxorubicin (DOX) encapsulated in polymeric nanoparticles (NPs) modified with targeting ligands against CD19 (CD19-DOX-NPs) can be delivered in a CD19-specific manner to leukemic cells. The CD19-DOX-NPs were internalized via receptor-mediated endocytosis and imparted cytotoxicity in a CD19-dependent manner in CD19-positive ALL cells. Leukemic mice treated with CD19-DOX-NPs survived significantly longer and manifested a higher degree of agility, indicating reduced apparent systemic toxicity during treatment compared to mice treated with free DOX. We suggest that targeted delivery of drugs used in childhood cancer treatment should improve therapeutic efficacy and reduce treatment-related side effects in children.
Subject(s)
Antigens, CD19/metabolism , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Drug Delivery Systems/methods , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Animals , Cell Line, Tumor , Cell Survival/drug effects , Doxorubicin/chemistry , Humans , Mice , Mice, Inbred BALB CABSTRACT
Na,K-ATPase is a hetero-oligomer of an α- and a ß-subunit. The α-subunit (Na,K-α) possesses the catalytic function, whereas the ß-subunit (Na,K-ß) has cell-cell adhesion function and is localized to the apical junctional complex in polarized epithelial cells. Earlier, we identified two distinct conserved motifs on the Na,K-ß(1) transmembrane domain that mediate protein-protein interactions: a glycine zipper motif involved in the cis homo-oligomerization of Na,K-ß(1) and a heptad repeat motif that is involved in the hetero-oligomeric interaction with Na,K-α(1). We now provide evidence that knockdown of Na,K-ß(1) prevents lumen formation and induces activation of extracellular regulated kinases 1 and 2 (ERK1/2) mediated by phosphatidylinositol 3-kinase in MDCK cells grown in three-dimensional collagen cultures. These cells sustained cell proliferation in an ERK1/2-dependent manner and did not show contact inhibition at high cell densities, as revealed by parental MDCK cells. This phenotype could be rescued by wild-type Na,K-ß(1) or heptad repeat motif mutant of Na,K-ß(1), but not by the glycine zipper motif mutant that abrogates Na,K-ß(1) cis homo-oligomerization. These studies suggest that Na,K-ß(1) cis homo-oligomerization rather than hetero-oligomerization with Na,K-α(1) is involved in epithelial lumen formation. The relevance of these findings to pre-neoplastic lumen filling in epithelial cancer is discussed.
Subject(s)
Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Cell Line , Cell Proliferation , Dogs , Immunoblotting , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein Multimerization/genetics , Protein Multimerization/physiology , Sodium-Potassium-Exchanging ATPase/chemistryABSTRACT
Nanotechnology approaches have tremendous potential for enhancing treatment efficacy with lower doses of chemotherapeutics. Nanoparticle (NP)-based drug delivery approaches are poorly developed for childhood leukemia. Dexamethasone (Dex) is one of the most common chemotherapeutic drugs used in the treatment of childhood leukemia. In this study, we encapsulated Dex in polymeric NPs and validated their antileukemic potential in vitro and in vivo. NPs with an average diameter of 110 nm were assembled from an amphiphilic block copolymer of poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals (ECT2). The blank NPs were nontoxic to cultured cells in vitro and to mice in vivo. Encapsulation of Dex into the NPs (Dex-NP) did not compromise the bioactivity of the drug. Dex-NPs induced glucocorticoid phosphorylation and showed cytotoxicity similar to the free Dex in leukemic cells. Studies using NPs labeled with fluorescent dyes revealed leukemic cell surface binding and internalization. In vivo biodistribution studies showed NP accumulation in the liver and spleen with subsequent clearance of the particles with time. In a preclinical model of leukemia, Dex-NPs significantly improved the quality of life and survival of mice as compared to the free drug. To our knowledge, this is the first report showing the efficacy of polymeric NPs to deliver Dex to potentially treat childhood leukemia and reveals that low doses of Dex should be sufficient for inducing cell death and improving survival.
Subject(s)
Dexamethasone/chemistry , Dexamethasone/therapeutic use , Leukemia/drug therapy , Nanomedicine/methods , Nanoparticles/chemistry , Polymers/chemistry , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Female , Humans , Liver/metabolism , Mice , Mice, Inbred BALB C , Phosphorylation/drug effects , Polyesters/chemistry , Polyethylene Glycols/chemistry , Spleen/metabolismABSTRACT
The phosphatase and tensin homolog (PTEN) is a tumor suppressor that is inactivated in many human cancers. PTEN loss has been associated with resistance to inhibitors of the epidermal growth factor receptor (EGFR), but the molecular basis of this resistance is unclear. It is believed that unopposed phosphatidylinositol-3-kinase (PI3K) activation through multiple receptor tyrosine kinases (RTKs) can relieve PTEN-deficient cancers from their "dependence" on EGFR or any other single RTK for survival. Here we report a distinct resistance mechanism whereby PTEN inactivation specifically raises EGFR activity by impairing the ligand-induced ubiquitylation and degradation of the activated receptor through destabilization of newly formed ubiquitin ligase Cbl complexes. PTEN-associated resistance to EGFR kinase inhibitors is phenocopied by expression of dominant negative Cbl and can be overcome by more complete EGFR kinase inhibition. PTEN inactivation does not confer resistance to inhibitors of the MET or PDGFRA kinase. Our study identifies a critical role for PTEN in EGFR signal termination and suggests that more potent EGFR inhibition should overcome resistance caused by PI3K pathway activation.
Subject(s)
ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , PTEN Phosphohydrolase/metabolism , Protein Kinase Inhibitors/pharmacology , Animals , Apoptosis , Cell Line , Enzyme Activation , Humans , Mice , Mice, Knockout , PTEN Phosphohydrolase/deficiency , PTEN Phosphohydrolase/genetics , Protein Binding , Proto-Oncogene Proteins c-cbl/metabolism , RNA Interference , Signal Transduction/drug effects , UbiquitinationABSTRACT
Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression.
Subject(s)
Adenocarcinoma/enzymology , Lung Neoplasms/enzymology , Nicotiana , Smoke/adverse effects , Sodium-Potassium-Exchanging ATPase/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Cell Line, Tumor , Disease Progression , Disease-Free Survival , Humans , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Neoplasm Grading , Reactive Oxygen Species/metabolism , Smoking/adverse effects , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Sodium-Potassium-Exchanging ATPase/biosynthesisABSTRACT
High levels of the soluble form of E-cadherin can be found in the serum of cancer patients and are associated with poor prognosis. Despite the possible predictive value of soluble E-cadherin, little is understood concerning its patho-physiological consequences in tumor progression. In this study, we show that soluble E-cadherin facilitates cell survival via functional interaction with cellular E-cadherin. Exposure of cells to a recombinant form of soluble E-cadherin, at a concentration found in cancer patient's serum, prevents apoptosis due to serum/growth factor withdrawal, and inhibits epithelial lumen formation, a process that requires apoptosis. Further, soluble E-cadherin-mediated cell survival involves activation of the epidermal growth factor receptor (EGFR) and EGFR-mediated activation of both phosphoinositide-3 kinase (PI3K)/AKT and ERK1/2 signaling pathways. These results are evidence of a complex functional interplay between EGFR and E-cadherin and also suggest that the presence of soluble E-cadherin in cancer patients' sera might have relevance to cell survival and tumor progression.
Subject(s)
Apoptosis/drug effects , Cadherins/pharmacology , ErbB Receptors/metabolism , Gene Expression Regulation/drug effects , Cadherins/metabolism , Cell Survival/drug effects , HEK293 Cells , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Oncogene Protein v-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction/drug effects , SolubilityABSTRACT
BACKGROUND: Tissue microarray (TMA) data are commonly used to validate the prognostic accuracy of tumor markers. For example, breast cancer TMA data have led to the identification of several promising prognostic markers of survival time. Several studies have shown that TMA data can also be used to cluster patients into clinically distinct groups. Here we use breast cancer TMA data to cluster patients into distinct prognostic groups. METHODS: We apply weighted correlation network analysis (WGCNA) to TMA data consisting of 26 putative tumor biomarkers measured on 82 breast cancer patients. Based on this analysis we identify three groups of patients with low (5.4%), moderate (22%) and high (50%) mortality rates, respectively. We then develop a simple threshold rule using a subset of three markers (p53, Na-KATPase-ß1, and TGF ß receptor II) that can approximately define these mortality groups. We compare the results of this correlation network analysis with results from a standard Cox regression analysis. RESULTS: We find that the rule-based grouping variable (referred to as WGCNA*) is an independent predictor of survival time. While WGCNA* is based on protein measurements (TMA data), it validated in two independent Affymetrix microarray gene expression data (which measure mRNA abundance). We find that the WGCNA patient groups differed by 35% from mortality groups defined by a more conventional stepwise Cox regression analysis approach. CONCLUSIONS: We show that correlation network methods, which are primarily used to analyze the relationships between gene products, are also useful for analyzing the relationships between patients and for defining distinct patient groups based on TMA data. We identify a rule based on three tumor markers for predicting breast cancer survival outcomes.
Subject(s)
Biomarkers, Tumor/biosynthesis , Breast Neoplasms/metabolism , Neoplasm Proteins/biosynthesis , Protein Serine-Threonine Kinases/biosynthesis , Receptors, Transforming Growth Factor beta/biosynthesis , Sodium-Potassium-Exchanging ATPase/biosynthesis , Tumor Suppressor Protein p53/biosynthesis , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Cluster Analysis , Female , Gene Expression Regulation, Neoplastic , Genes, p53 , Humans , Neoplasm Proteins/genetics , Prognosis , Proportional Hazards Models , Protein Array Analysis , Protein Serine-Threonine Kinases/genetics , Receptor, Transforming Growth Factor-beta Type II , Receptors, Transforming Growth Factor beta/genetics , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
BACKGROUND: Childhood cancer remains the leading cause of disease-related mortality for children. Whereas, improvement in care has dramatically increased survival, the risk factors remain to be fully understood. The increasing incidence of childhood cancer in Florida may be associated with possible cancer clusters. We aimed, in this study, to identify and confirm possible childhood cancer clusters and their subtypes in the state of Florida. METHODS: We conducted purely spatial and space-time analyzes to assess any evidence of childhood malignancy clusters in the state of Florida using SaTScan. Data from the Florida Association of Pediatric Tumor Programs (FAPTP) for the period 2000-2007 were used in this analysis. RESULTS: In the purely spatial analysis, the relative risks (RR) of overall childhood cancer persisted after controlling for confounding factors in south Florida (SF) (RR = 1.36, P = 0.001) and northeastern Florida (NEF) (RR = 1.30, P = 0.01). Likewise, in the space-time analysis, there was a statistically significant increase in cancer rates in SF (RR = 1.52, P = 0.001) between 2006 and 2007. The purely spatial analysis of the cancer subtypes indicated a statistically significant increase in the rate of leukemia and brain/CNS cancers in both SF and NEF, P < 0.05. The space-time analysis indicated a statistically significant sizable increase in brain/CNS tumors (RR = 2.25, P = 0.02) for 2006-2007. CONCLUSIONS: There is evidence of spatial and space-time childhood cancer clustering in SF and NEF. This evidence is suggestive of the presence of possible predisposing factors in these cluster regions. Therefore, further study is needed to investigate these potential risk factors.
Subject(s)
Neoplasms/epidemiology , Child , Female , Florida/epidemiology , Humans , Incidence , Male , Space-Time ClusteringABSTRACT
Na,K-ATPase is composed of two essential alpha- and beta-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its alpha(1), alpha(3) and beta(1) isoforms are reduced in the failing human heart. The catalytic alpha-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase beta(1)-subunit (Na,K-beta(1)) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-beta(1) gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-beta(1) gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13-14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-beta(1) KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-beta(1) KO in older mice. Further, we show that intact hearts of Na,K-beta(1) KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-beta(1) plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.
Subject(s)
Gene Deletion , Myocardial Contraction/drug effects , Myocardium/enzymology , Ouabain/pharmacology , Protein Subunits/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Aging/drug effects , Animals , Calcium Signaling/drug effects , Cardiomegaly/enzymology , Cardiomegaly/physiopathology , Cell Separation , Heart Function Tests , Immunoblotting , Mice , Mice, Knockout , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Organ Specificity/drug effects , Pressure , Sodium-Calcium Exchanger/metabolismABSTRACT
Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.
Subject(s)
Tight Junctions/physiology , Animals , Epithelial Cells/physiology , Epithelial Cells/ultrastructure , Humans , Ion Channels/physiology , Membrane Proteins/chemistry , Membrane Proteins/physiology , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/physiology , Models, Biological , Models, Molecular , Paracrine Communication , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/physiology , Tight Junctions/ultrastructureABSTRACT
Loss of alpha-catenin is one of the characteristics of prostate cancer. The catenins (alpha and beta) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of beta-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where beta-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, beta-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of beta-catenin invariably are altered in cancer. Although a wealth of information is available about beta-catenin deregulation during oncogenesis, much less is known about how or whether alpha-catenin regulates beta-catenin functions. In this study, we show that alpha-catenin acts as a switch regulating the cell-cell adhesion and proliferation functions of beta-catenin. In alpha-catenin-null prostate cancer cells, reexpression of alpha-catenin increased cell-cell adhesion and decreased beta-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of beta-catenin is a major mechanism for decreased beta-catenin interaction with E-cadherin in alpha-catenin-null cells. alpha-Catenin attenuated the effect of Src phosphorylation by increasing beta-catenin association with E-cadherin. We also show that alpha-catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that alpha-catenin is a key regulator of beta-catenin transcriptional activity and that the status of alpha-catenin expression in tumor tissues might have prognostic value for Src targeted therapy.
Subject(s)
Oncogene Proteins/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , Signal Transduction , alpha Catenin/metabolism , beta Catenin/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cell Proliferation , Cyclin D1/metabolism , Gene Expression Regulation, Neoplastic , Humans , Intercellular Junctions/metabolism , Intercellular Junctions/ultrastructure , Male , Phosphorylation , Transcription, Genetic , beta Catenin/geneticsABSTRACT
Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in advanced and metastatic prostate cancers. The pathologic consequence of elevated PSMA expression in not known. Here, we report that PSMA is localized to a membrane compartment in the vicinity of mitotic spindle poles and associates with the anaphase-promoting complex (APC). PSMA-expressing cells prematurely degrade cyclin B and exit mitosis due to increased APC activity and incomplete inactivation of APC by the spindle assembly checkpoint. Further, expression of PSMA in a karyotypically stable cell line induces aneuploidy. Thus, these findings provide the first evidence that PSMA has a causal role in the induction of aneuploidy and might play an etiologic role in the progression of prostate cancer.
Subject(s)
Chromosomal Instability , Prostate-Specific Antigen/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Anaphase-Promoting Complex-Cyclosome , Animals , Cell Cycle/drug effects , Cell Line, Tumor , Centrosome/drug effects , Centrosome/ultrastructure , Chromosomal Instability/drug effects , Cyclin B/metabolism , Cyclin B1 , Dogs , Humans , Nocodazole/pharmacology , Prostate-Specific Antigen/ultrastructure , Protein Binding/drug effects , Protein Transport/drug effects , Spindle Apparatus/metabolismABSTRACT
Na,K-ATPase is a hetero-oligomer of alpha and beta-subunits. The Na,K-ATPase beta-subunit (Na,K-beta) is involved in both the regulation of ion transport activity, and in cell-cell adhesion. By structure prediction and evolutionary analysis, we identified two distinct faces on the Na,K-beta transmembrane domain (TMD) that could mediate protein-protein interactions: a glycine zipper motif and a conserved heptad repeat. Here, we show that the heptad repeat face is involved in the hetero-oligomeric interaction of Na,K-beta with Na,K-alpha, and the glycine zipper face is involved in the homo-oligomerization of Na,K-beta. Point mutations in the heptad repeat motif reduced Na,K-beta binding to Na,K-alpha, and Na,K-ATPase activity. Na,K-beta TMD homo-oligomerized in biological membranes, and mutation of the glycine zipper motif affected oligomerization and cell-cell adhesion. These results provide a structural basis for understanding how Na,K-beta links ion transport and cell-cell adhesion.
Subject(s)
Models, Molecular , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/metabolism , Amino Acid Sequence , Animals , Cell Aggregation , Cell Membrane/enzymology , Dogs , Glycine/genetics , Leucine/genetics , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , Repetitive Sequences, Amino AcidABSTRACT
PURPOSE: Na,K-adenosine triphosphatase, which is composed of a catalytic alpha-subunit and a regulatory beta-subunit, generates an electrochemical gradient across the plasma membrane. Previous studies demonstrated altered Na,K-adenosine triphosphatase subunit expression in renal clear cell carcinoma and an association of subunit levels with the prediction of recurrent bladder cancer. We determined the clinical association of protein expression patterns of the Na,K-adenosine triphosphatase alpha1 and beta1-subunits in renal clear cell carcinoma using tissue microarrays with linked clinicopathological data. MATERIALS AND METHODS: The UCLA kidney cancer tissue microarray was used to investigate the protein expression of Na,K-adenosine triphosphatase alpha1 and beta1-subunits by immunohistochemistry in 342 patients with renal clear cell carcinoma who were treated with radical nephrectomy. Of these patients clinical outcomes studies were performed in 317. The resultant expression reactivity was correlated with clinicopathological variables. RESULTS: We found that the alpha1-subunit was a significant and independent predictor of disease specific death from renal clear cell carcinoma on multivariate Cox proportional hazards analysis that included established prognostic factors Eastern Cooperative Oncology Group performance status, pT status, metastasis status and tumor grade. Significance was found when examining all patients with clear cell renal cell carcinoma as well as patient substrata with low or high grade tumors and localized or metastatic disease, suggesting that the Na,K-adenosine triphosphatase alpha1-subunit could be used as a new prognosticator for disease specific death from renal clear cell carcinoma. CONCLUSIONS: These results suggest that Na,K-adenosine triphosphatase alpha1-subunit expression patterns may be a useful clinical prognosticator for renal clear cell carcinoma. The Na,K-adenosine triphosphatase beta1-subunit was not found to be a useful prognosticator in this setting.
Subject(s)
Biomarkers, Tumor/biosynthesis , Carcinoma, Renal Cell/enzymology , Carcinoma, Renal Cell/mortality , Kidney Neoplasms/enzymology , Kidney Neoplasms/mortality , Sodium-Potassium-Exchanging ATPase/biosynthesis , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Predictive Value of Tests , Survival RateABSTRACT
The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.
Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Sodium-Potassium-Exchanging ATPase/physiology , Actins/chemistry , Actins/metabolism , Animals , Annexin A2/chemistry , Annexin A2/genetics , Cell Line , Cell Movement , Chromatography, Liquid , Chromones/pharmacology , Cloning, Molecular , Cytoplasm/metabolism , Cytoskeleton , Dogs , Epithelial Cells/cytology , Glutathione Transferase/metabolism , Immunoblotting , Immunoprecipitation , Ions , Mass Spectrometry , Microscopy, Confocal , Microscopy, Fluorescence , Models, Biological , Morpholines/pharmacology , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Phalloidine/pharmacology , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Signal Transduction , Sodium-Potassium-Exchanging ATPase/chemistry , Tight Junctions , rac1 GTP-Binding Protein/metabolismABSTRACT
Fibrous proteins found in the natural extracellular matrix (ECM) function as host substrates for migration and growth of endogenous cells during wound healing and tissue repair processes. Although various fibrous scaffolds have been developed to recapitulate the microstructures of the native ECM, facile synthesis of hydrogel microfibers that are mechanically robust and biologically active have been elusive. Described herein is the use of interfacial bioorthogonal polymerization to create hydrogel-based microfibrous scaffolds via tetrazine ligation. Combination of a trifunctional strained trans-cyclooctene monomer and a difunctional s-tetrazine monomer at the oil-water interface led to the formation of microfibers that were stable under cell culture conditions. The bioorthogonal nature of the synthesis allows for direct incorporation of tetrazine-conjugated peptides or proteins with site-selectively, genetically encoded tetrazines. The microfibers provide physical guidance and biochemical signals to promote the attachment, division and migration of fibroblasts. Mechanistic investigations revealed that fiber-guided cell migration was both F-actin and microtubule-dependent, confirming contact guidance by the microfibers. Prolonged culture of fibroblasts in the presence of an isolated microfiber resulted in the formation of a multilayered cell sheet wrapping around the fiber core. A fibrous mesh provided a 3D template to promote cell infiltration and tissue-like growth. Overall, the bioorthogonal approach led to the straightforward synthesis of crosslinked hydrogel microfibers that can potentially be used as instructive materials for tissue repair and regeneration.
Subject(s)
Hydrogels/chemistry , Animals , Cell Culture Techniques , Cell Movement/physiology , Fibroblasts/cytology , Humans , Peptides/chemistry , Polymerization , Proteins/chemistry , Tissue Scaffolds/chemistry , Wound Healing/physiologyABSTRACT
Chemical modification of engineered microenvironments surrounding living cells represents a means for directing cellular behaviors through cell-matrix interactions. Presented here is a temporally controlled method for modulating the properties of biomimetic, synthetic extracellular matrices (ECM) during live cell culture employing the rapid, bioorthogonal tetrazine ligation with trans-cyclooctene (TCO) dienophiles. This approach is diffusion-controlled, cytocompatible, and does not rely on light, catalysts, or other external triggers. Human bone-marrow-derived mesenchymal stem cells (hMSCs) were initially entrapped in a hydrogel prepared using hyaluronic acid carrying sulfhydryl groups (HA-SH) and a hydrophilic polymer bearing both acrylate and tetrazine groups (POM-AT). Inclusion of a matrix metalloprotease (MMP)-degradable peptidic cross-linker enabled hMSC-mediated remodeling of the synthetic environment. The resultant network displayed dangling tetrazine groups for subsequent conjugation with TCO derivatives. Two days later, the stiffness of the matrix was increased by adding chemically modified HA carrying multiple copies of TCO (HA-TCO) to the hMSC growth media surrounding the cell-laden gel construct. In response, cells developed small processes radially around the cell body without a significant alteration of the overall shape. By contrast, modification of the 3D matrix with a TCO-tagged cell-adhesive motif caused the resident cells to undergo significant actin polymerization, changing from a rounded shape to spindle morphology with long cellular processes. After additional 7 days of culture in the growth media, quantitative analysis showed that, at the mRNA level, RGD tagging upregulated cellular expression of MMP1, but downregulated the expression of collagen I/III and tenascin C. RGD tagging, however, was not sufficient to induce the classic osteoblastic, chondrogenic, adipogenic, or fibroblastic/myofibroblastic differentiation. The modular approach allows facile manipulation of synthetic ECM to modulate cell behavior, thus potentially applicable to the engineering of functional tissues or tissue models.
Subject(s)
Stem Cells , Cell Culture Techniques , Cell Differentiation , Chondrogenesis , Extracellular Matrix , Humans , Hydrogels , Mesenchymal Stem CellsABSTRACT
Prostate-specific membrane antigen (PSMA) is an integral membrane glycoprotein expressed in prostatic epithelia and is being evaluated as a therapeutic target in prostate cancer. It undergoes constitutive receptor-mediated endocytosis via clathrin-coated pits, which is enhanced in the presence of monoclonal antibodies directed against it. We describe distinct interactions of PSMA with clathrin and the clathrin adaptor protein-2 (AP-2) complex, two components of clathrin-coated pits. The intracellular N-terminal domain of PSMA interacts with the N-terminal globular domain of clathrin heavy chain. Deletion analysis revealed an important determinant of this interaction residing within the proximal portion of the clathrin heavy chain N-terminal domain (amino acids 1-85) distinct from the clathrin binding sites of other known clathrin-binding proteins. Furthermore, PSMA interacts with the ear domain of alpha-adaptin (an AP-2 subunit), and a glutamic acid residue at position 7 in the cytoplasmic tail of PSMA is essential for this interaction. These data indicate that PSMA exhibits a high affinity, specific association with the clathrin-based endocytic machinery by distinct interactions with both clathrin and AP-2. Thus, although PSMA is a new member of the dual AP and clathrin binding proteins, its alpha-adaptin and clathrin heavy chain binding determinants are distinct from those of other members.