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1.  Dishonorable Discharge: The Oncogenic Roles of Cleaved E-cadherin Fragments 
Cancer Research  2012;72(12):2917-2923.
Loss of intercellular adhesion by E-cadherin is a fundamental change that occurs during the progression of cancer to invasive disease as strong cell-cell interaction represents a major barrier to cancer cell mobility. However, some aggressive carcinomas retain characteristics of differentiated epithelial cells including E-cadherin expression. Emerging evidence indicates that proteolysis of E-cadherin generates fragments that promote tumor growth, survival, and motility, suggesting that E-cadherin cleavage converts this tumor suppressor into an oncogenic factor. In this review we discuss the emerging roles of cleaved E-cadherin fragments as modulators of cancer progression and explore the translational and clinical implications of this research.
doi:10.1158/0008-5472.CAN-11-3498
PMCID: PMC3377785  PMID: 22659456
Signal transduction; Tumor promotion and progression; Cell adhesion; Cell-cell interactions; Growth factors: structure and function; Receptors: structure and function; Cell adhesion/cell-cell interactions in apoptosis; Survival factors; Cell motility and migration; Tumor markers and detection of metastasis
2.  Evidence for a potential tumor suppressor role for the Na,K-ATPase ß1-subunit 
Histology and histopathology  2008;23(4):459-467.
Summary
The Na,K-ATPase, consisting of two essential subunits (α, ß), plays a critical role in the regulation of ion homeostasis in mammalian cells. Recent studies indicate that reduced expression of the ß1 isoform (NaK-ß1) is commonly observed in carcinoma and is associated with events involved in cancer progression. In this study, we present evidence that repletion of NaK-ß1 in Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK), a highly tumorigenic cell line, inhibits anchorage independent growth and suppresses tumor formation in immunocompromised mice. Additionally, using an in vitro cell-cell aggregation assay, we showed that cell aggregates of NaK-ß1 subunit expressing MSV-MDCK cells have reduced extracellular regulated kinase (ERK) 1/2 activity compared with parental MSV-MDCK cells. Finally, using immunohistochemistry and fully quantitative image analysis approaches, we showed that the levels of phosphorylated ERK 1/2 are inversely correlated to the NaK-ß1 levels in the tumors. These findings reveal for the first time that NaK-ß1 has a potential tumor-suppressor function in epithelial cells.
PMCID: PMC2779022  PMID: 18228203
Tumorigenicity; Cell adhesion; Na; K-ATPase ß-subunit; ERK 1/2
3.  Protein expression based multimarker analysis of breast cancer samples 
BMC Cancer  2011;11:230.
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-β1, and TGF β 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.
doi:10.1186/1471-2407-11-230
PMCID: PMC3142534  PMID: 21651811
Tissue microarray; breast cancer; tumor marker; prognostic marker; WGCNA
4.  Na, K-ATPase subunits as markers for epithelial-mesenchymal transition in cancer and fibrosis 
Molecular cancer therapeutics  2010;9(6):1515-1524.
Epithelial-to-mesenchymal transition (EMT) is an important developmental process, participates in tissue repair and occurs during pathological processes of tumor invasiveness, metastasis and tissue fibrosis. The molecular mechanisms leading to EMT are poorly understood. While it is well documented that transforming growth factor (TGF)-β plays a central role in the induction of EMT, the targets of TGF-β signaling are poorly defined. We have shown earlier that Na,K-ATPase β1-subunit levels are highly reduced in poorly differentiated kidney carcinoma cells in culture and in patients’ tumor samples. In this study, we provide evidence that Na,K-ATPase is a new target of TGF-β1-mediated EMT in renal epithelial cells, a model system used in studies of both cancer progression and fibrosis. We show that following treatment with TGF-β1 the surface expression of the β1-subunit of Na,K-ATPase is reduced, prior to well-characterized EMT markers and is associated with the acquisition of a mesenchymal phenotype. RNAi mediated knockdown confirmed the specific involvement of the Na,K-ATPase β1-subunit in the loss of the epithelial phenotype and exogenous over-expression of the Na,K-ATPase β1-subunit attenuated TGF-β1-mediated EMT. We further show that both Na,K-ATPase α- and β-subunit levels are highly reduced in renal fibrotic tissues. These findings for the first time reveal that Na,K-ATPase is a target of TGF-β1-mediated EMT and is associated with the progression of EMT in both cancer and fibrosis.
doi:10.1158/1535-7163.MCT-09-0832
PMCID: PMC2884047  PMID: 20501797
Transforming growth factor (TGF)-β1; Na,K-ATPase; Epithelial-to-mesenchymal transition (EMT); cancer; fibrosis
5.  Dysfunction of ouabain-induced cardiac contractility in mice with heart-specific ablation of Na,K-ATPase β1-subunit 
Na,K-ATPase is composed of two essential α- and β-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its α1, α3 and β1 isoforms are reduced in the failing human heart. The catalytic α-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 β1-subunit (Na,K-β1) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-β1 gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-β1 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-β1 KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-β1 KO in older mice. Further, we show that intact hearts of Na,K-β1 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-β1 plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.
doi:10.1016/j.yjmcc.2009.07.018
PMCID: PMC2749246  PMID: 19683723
Na,K-ATPase; Na,K-ATPase β1-subunit; hypertrophy; contractility; TAC; ouabain; cardiomyocytes; NCX1
6.  Expression of Na,K-ATPase-β1 subunit increases uptake and sensitizes carcinoma cells to oxaliplatin 
Cancer Chemotherapy and Pharmacology  2009;64(6):1187-1194.
Purpose
The ovarian carcinoma subline A2780/C10B (C10B) is an oxaliplatin resistant clone derived from the human ovarian carcinoma cell line A2780. The C10B cells are characterized by mesenchymal phenotype, decreased platinum uptake and increased glutathione levels (Hector et al. in Cancer Lett 245:195–204, 2007; Varma et al. in Oncol Rep 14:925–932, 2005). Na,K-ATPase-β subunit (Na,K-β1) functions as a cell–cell adhesion molecule in epithelial cells and is reduced in a variety of carcinoma cells that show mesenchymal phenotype. The purpose of this study is to evaluate the relationship between Na,K-β expression and sensitivity to oxaliplatin.
Methods
Cell lines used include A2780, C10B, C10B transfected with Na,K-β1 (C10B-Na,K-β) and a canine kidney carcinoma cell line MSV-MDCK also transfected with Na,K-β1 (MSV-MDCK-β subunit). Cytotoxicity studies were performed by sulforhodamine-blue assay. The Na,K-α1 and Na,K-β1 subunit localization and expression were by immunofluorescence microscopy and Western blot analysis. Platinum accumulation measurements were by atomic absorption spectrophotometry.
Results
C10B cells express highly reduced levels of Na,K-β1 subunit. Exogenous expression of Na,K-β1 increased platinum accumulation and sensitized C10B cells to oxaliplatin. The pharmacological inhibitor of Na,K-ATPase ouabain did not alter the oxaliplatin accumulation indicating that Na,K-β1 sensitizes cells in a Na,K-ATPase enzyme activity independent manner. These findings were also confirmed in MSV-MDCK-β subunit cells.
Conclusions
This study for the first time reveals that reduced expression of the Na,K-β1 protein is associated with oxaliplatin resistance in cancer cells and demonstrates a novel role for this protein in sensitizing the cells to oxaliplatin. This study suggests a potentially important role for Na,K-β1 in both prognosis and therapy of oxaliplatin resistant malignancies.
doi:10.1007/s00280-009-0985-x
PMCID: PMC2728910  PMID: 19322565
7.  Prostate specific membrane antigen associates with anaphase promoting complex and induces chromosomal instability 
Molecular cancer therapeutics  2008;7(7):2142-2151.
Prostate specific membrane antigen (PSMA) is a transmembrane protein highly expressed in advanced and metastatic prostate cancers. The pathological 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 etiological role in the progression of prostate cancer.
doi:10.1158/1535-7163.MCT-08-0005
PMCID: PMC2548318  PMID: 18645024
Prostate specific membrane antigen; cell cycle; anaphase promoting complex; aneuploidy; prostate cancer
8.  α-catenin overrides Src-dependent activation of β-catenin oncogenic signaling 
Molecular cancer therapeutics  2008;7(6):1386-1397.
Loss of α-catenin is one of the characteristics of prostate cancer. The catenins (α, β) associated with E-cadherin play a critical role in the regulation of cell-cell adhesion. Tyrosine phosphorylation of β-catenin dissociates it from E-cadherin and facilitates its entry into the nucleus, where β-catenin acts as a transcriptional activator inducing genes involved in cell proliferation. Thus, β-catenin regulates cell-cell adhesion and cell proliferation. Mechanisms controlling the balance between these functions of β-catenin invariably are altered in cancer. Although a wealth of information is available about β-catenin deregulation during oncogenesis, much less is known about how or whether α-catenin regulates β-catenin functions. In this study, we show that α-catenin acts as a switch regulating β-catenin’s cell-cell adhesion and proliferation functions. In α-catenin null prostate cancer cells, re-expression of α-catenin increased cell-cell adhesion and decreased β-catenin transcriptional activity, cyclin D1 levels, and cell proliferation. Further, Src-mediated tyrosine phosphorylation of β-catenin is a major mechanism for decreased β-catenin interaction with E-cadherin in α-catenin null cells. α-catenin attenuated the effect of Src phosphorylation by increasing β-catenin association with E-cadherin. We also show that α-catenin increases the sensitivity of prostate cancer cells to a Src inhibitor in suppressing cell proliferation. This study reveals for the first time that α-catenin is a key regulator of β-catenin transcriptional activity and that the status of α-catenin expression in tumor tissues might have prognostic value for Src targeted therapy.
doi:10.1158/1535-7163.MCT-07-2029
PMCID: PMC2527861  PMID: 18566211
catenin; prostate cancer; adherens junction; src; TCF/LEF
9.  Janus Model of The Na,K-ATPase β-subunit Transmembrane Domain: Distinct Faces Mediate α /β Assembly and β-β Homo-Oligomerization 
Journal of molecular biology  2006;365(3):706-714.
Summary
Na,K-ATPase is a hetero-oligomer of α- and β-subunits. The Na,K-ATPase β-subunit (Na,K-β ) 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-β 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-β with Na,K-α , and the glycine zipper face is involved in the homo-oligomerization of Na,K-β . Point mutations in the heptad repeat motif reduced Na,K-β binding to Na,K-α , and Na,K-ATPase activity. Na,K-β 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-β links ion transport and cell-cell adhesion.
doi:10.1016/j.jmb.2006.10.029
PMCID: PMC2459552  PMID: 17078968
transmembrane domain; heptad repeat motif; GxxxG; glycine zipper; Na; K-ATPase; cell adhesion
10.  Novel Role for Na,K-ATPase in Phosphatidylinositol 3-Kinase Signaling and Suppression of Cell Motility 
Molecular Biology of the Cell  2005;16(3):1082-1094.
The Na,K-ATPase, consisting of α- and β-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 β1-subunit (Na,K-β) 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-β reduces cell motility remained elusive. Here, we demonstrate that Na,K-β localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase α1-subunit (Na,K-α) and Na,K-β with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-α associates with the regulatory subunit of PI3-kinase and Na,K-β 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.
doi:10.1091/mbc.E04-05-0427
PMCID: PMC551475  PMID: 15616195
11.  Na,K-ATPase β1-Subunit Increases the Translation Efficiency of the α1-Subunit in MSV-MDCK Cells 
Molecular Biology of the Cell  2004;15(7):3224-3232.
The Na,K-ATPase consists of an α- and β-subunit. Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK) express low levels of Na,K-ATPase β1-subunit. Ectopic expression of Na,K-ATPase β1-subunit in these cells increased the protein levels of the α1-subunit of Na,K-ATPase. This increase was not due to altered transcription of the α1-subunit gene or half-life of the α1-subunit protein because both α1-subunit mRNA levels and half-life of the α1-subunit protein were comparable in MSV-MDCK and β1-subunit expressing MSV-MDCK cells. However, short pulse labeling revealed that the initial translation rate of the α1-subunit in β1-subunit expressing MSV-MDCK cells was six- to sevenfold higher compared with MSV-MDCK cells. The increased translation was specific to α1-subunit because translation rates of occludin and β-catenin, membrane and cytosolic proteins, respectively, were not altered. In vitro cotranslation/translocation experiments using rabbit reticulocyte lysate and rough microsomes revealed that the α1-subunit mRNA is more efficiently translated in the presence of β1-subunit. Furthermore, sucrose density gradient analysis revealed significantly more α1-subunit transcript associated with the polysomal fraction in β1-subunit expressing MSV-MDCK cells compared with MSV-MDCK cells, indicating that in mammalian cells the Na,K-ATPase β1-subunit is involved in facilitating the translation of the α1-subunit mRNA in the endoplasmic reticulum.
doi:10.1091/mbc.E04-03-0222
PMCID: PMC452578  PMID: 15133131
12.  Repression of Na,K-ATPase β1-Subunit by the Transcription Factor Snail in Carcinoma 
Molecular Biology of the Cell  2004;15(3):1364-1373.
The Na,K-ATPase consists of two essential α- and β-subunits and regulates the intracellular Na+ and K+ homeostasis. Although the α-subunit contains the catalytic activity, it is not active without functional β-subunit. Here, we report that poorly differentiated carcinoma cell lines derived from colon, breast, kidney, and pancreas show reduced expression of the Na,K-ATPase β1-subunit. Decreased expression of β1-subunit in poorly differentiated carcinoma cell lines correlated with increased expression of the transcription factor Snail known to down-regulate E-cadherin. Ectopic expression of Snail in well-differentiated epithelial cell lines reduced the protein levels of E-cadherin and β1-subunit and induced a mesenchymal phenotype. Reduction of Snail expression in a poorly differentiated carcinoma cell line by RNA interference increased the levels of Na,K-ATPase β1-subunit. Furthermore, Snail binds to a noncanonical E-box in the Na,K-ATPase β1-subunit promoter and suppresses its promoter activity. These results suggest that down-regulation of Na,K-ATPase β1-subunit and E-cadherin by Snail are associated with events leading to epithelial to mesenchymal transition.
doi:10.1091/mbc.E03-09-0646
PMCID: PMC363145  PMID: 14699059
13.  A Novel Cytoplasmic Tail MXXXL Motif Mediates the Internalization of Prostate-specific Membrane Antigen 
Molecular Biology of the Cell  2003;14(12):4835-4845.
Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the α-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative μ2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.
doi:10.1091/mbc.E02-11-0731
PMCID: PMC284788  PMID: 14528023
14.  Na,K-ATPase Activity Is Required for Formation of Tight Junctions, Desmosomes, and Induction of Polarity in Epithelial Cells 
Molecular Biology of the Cell  2001;12(12):3717-3732.
Na,K-ATPase is a key enzyme that regulates a variety of transport functions in epithelial cells. In this study, we demonstrate a role for Na,K-ATPase in the formation of tight junctions, desmosomes, and epithelial polarity with the use of the calcium switch model in Madin-Darby canine kidney cells. Inhibition of Na,K-ATPase either by ouabain or potassium depletion prevented the formation of tight junctions and desmosomes and the cells remained nonpolarized. The formation of bundled stress fibers that appeared transiently in control cells was largely inhibited in ouabain-treated or potassium-depleted cells. Failure to form stress fibers correlated with a large reduction of RhoA GTPase activity in Na,K-ATPase-inhibited cells. In cells overexpressing wild-type RhoA GTPase, Na,K-ATPase inhibition did not affect the formation of stress fibers, tight junctions, or desmosomes, and epithelial polarity developed normally, suggesting that RhoA GTPase is an essential component downstream of Na,K-ATPase-mediated regulation of these junctions. The effects of Na,K-ATPase inhibition were mimicked by treatment with the sodium ionophore gramicidin and were correlated with the increased intracellular sodium levels. Furthermore, ouabain treatment under sodium-free condition did not affect the formation of junctions and epithelial polarity, suggesting that the intracellular Na+ homeostasis plays a crucial role in generation of the polarized phenotype of epithelial cells. These results thus demonstrate that the Na,K-ATPase activity plays an important role in regulating both the structure and function of polarized epithelial cells.
PMCID: PMC60750  PMID: 11739775
15.  Na,K-ATPase β-Subunit Is Required for Epithelial Polarization, Suppression of Invasion, and Cell Motility 
Molecular Biology of the Cell  2001;12(2):279-295.
The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an α- and β-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin–mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and β1-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase β1-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin–mediated cell-cell adhesion requires the Na,K-ATPase β-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the β1-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells.
PMCID: PMC30943  PMID: 11179415

Results 1-15 (15)