Resistance to platinum-based chemotherapy is the major barrier to treating epithelial ovarian cancer. To improve patient outcomes, it is critical to identify the underlying mechanisms that promote platinum resistance. Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells, but are unable to eliminate cancer initiating cells (CIC). To date, the relevant pathways that regulate ovarian CICs remain elusive. Several correlative studies have shown that Wnt/β-catenin pathway activation is associated with poor outcomes in patients with high-grade serous ovarian cancer (HGSOC). However, the functional relevance of these findings remain to be delineated. We have uncovered that Wnt/β-catenin pathway activation is a critical driver of HGSOC chemotherapy resistance, and targeted inhibition of this pathway, which eliminates CICs, represents a novel and effective treatment for chemoresistant HGSOC. Here we show that Wnt/β-catenin signaling is activated in ovarian CICs, and targeted inhibition of β-catenin potently sensitized cells to cisplatin and decreased CIC tumor sphere formation. Furthermore, the Wnt/β-catenin specific inhibitor iCG-001 potently sensitized cells to cisplatin and decreased stem-cell frequency in platinum resistant cells. Taken together, our data is the first report providing evidence that the Wnt/β-catenin signaling pathway maintains stem-like properties and drug resistance of primary HGSOC PDX derived platinum resistant models, and therapeutic targeting of this pathway with iCG-001/PRI-724, which has been shown to be well tolerated in Phase I trials, may be an effective treatment option. Epithelial ovarian cancer [EOC] is the most lethal gynecologic malignancy and is the fifth leading cause of cancer deaths in women . This yeCisplatin treatment of ovarian tumors is associated with an increase in stem cell markers in the residual tumors, and accordingly, ovarian tumors that are chemo-resistant are reported to have increased expression of CIC markers , 6-9. EvCanonical Wnt signaling, mediated through β-catenin, is identified as a critical regulator of chemo-resistance and CIC phenotype in many tumors, and inhibition of Wnt/β-catenin signaling is reported to increase sensitivity to chemotherapeutic agents in cancers -14. Howe3 tumor implants were grafted subcutaneously into nu/nu mice and propagated. Once excised, pathological comparison between the primary patient tumor and the PDX tumor were performed  models have been utilized to conserve original tumor characteristics, such as heterogeneous histology, clinical biomolecular signature, malignant phenotypes and genotypes. Therefore, in order to assess the role of the Wnt/β-catenin pathway in regulating platinum resistance, we first examined the activity of this pathway in PDX models that display varying sensitivity to platinum-based therapies. In order to generate PDX models, chemotherapy-naive tumors were removed from patients diagnosed with HGSOC and ∼2mmerformed . In ordee Figure . In addio Figure . Upon coo Figure . Expresso Figure . Furthero Figure .in-vitro in the presence of cisplatin in order to select for cells that have acquired platinum resistance [which is henceforth referred to as CP10]. Cellular viability assays revealed that CP10 was significantly more resistant to cisplatin, in comparison to the parental OV81.2    Figure  and form) Figure . This tu) Figure  indicatin Figure  resultedn Figure  and robun Figure . Decreass Figure . Since d) Figure , suggesthigh and TOP-eGFP low cell populations from ALDHpos CICs by FACS and studied the differences in chemo-resistance between these two populations   Figure  and exhi) Figure . CollectSince our results showed that Wnt/β-catenin activity is a critical driver of chemo-resistance and stem-like properties in ovarian tumor cells, we next explored the possibility of therapeutic targeting of this pathway to overcome platinum resistance. Wnt/β-catenin inhibitors are reported to be effective in various tumor models and are being tested for their ability to target CICs , 14, 34.pos ovarian CICs by ∼20 fold (∼1:2.3 to 1:46), showing that therapeutic targeting of β-catenin could decrease stem-like properties in ovarian CICs   Figure . CollectCurrent platinum-based treatment regimens in ovarian cancer are initially very effective at decreasing tumor burden because of reduction in tumor cells that are differentiated. However, CICs are known to be platinum-resistant and thus constitute one of the root causes of chemotherapy failure and tumor relapse. The absence of CIC-targeted therapies in ovarian cancer can be mainly attributed to a lack of in-depth understanding of the mechanisms driving and maintaining the stem-like properties in these cells. Signaling pathways like Notch have been implicated in regulating chemo-resistance in ovarian CICs, and targeting these cells by gamma secretase inhibitors [GSI] has been reported to increase response to platinum , highligin vivo and in vitro models. Specifically, we show that platinum-resistant PDX mouse models of HGSOC and primary platinum-resistant ovarian tumor cells have dramatically higher Wnt activity compared to matched platinum-sensitive cells. Our results support the involvement of Wnt/β-catenin signaling in both intrinsic and acquired platinum chemo-resistance. Wnt reporter activity was up-regulated in both the intrinsic model (OV145 PDX) and acquired model (OV81 PDX derived CP10) of platinum resistance. Up-regulation of Wnt ligand WNT5A was similar to both intrinsic and acquired models of platinum resistance, suggesting that the pathway is highly activated in both models of resistance.Numerous studies, including TCGA data analysis, has reported altered Wnt/β-catenin components in HGSOC, and correlative studies have shown that activation of this pathway is associated with poor patient outcomes , 17, 20.Expression of the Wnt receptor FZD1 and the Wnt feedback component DKK2 correlated with Wnt/β-catenin activity and response to platinum in CP10 and CP70 models. Interestingly, the analysis of several publically available ovarian cancer cohorts revealed that FZD1 and DKK2 expression correlate with poor survival  suggestiSpecific modulation of the Wnt/β-catenin pathway, either by β-catenin overexpression or knockdown, directly correlated with response to platinum-based therapy. β-catenin knockdown reversed platinum resistance and decreased stem cell frequency, which was associated with a decrease in the expression of stem cell markers like LEF-1 and LGR5. Intriguingly, these two markers were recently shown to be highly expressed in the ovarian stem cell niche , and LGRpos ovarian CIC population there exists a sub-population of TOP-eGFPhigh (Wnt-active) cells that are chemo-resistant and have higher expression of CIC markers, suggesting that Wnt activity could be critical in maintaining platinum resistance in ovarian CICs. Since ALDH1 is identified to be a direct target of β-catenin, and because we also saw a decrease in ALDH1 expression upon β-catenin knockdown, it could be speculated that the stem-like properties in ALDHpos CICs in ovarian cancers could indeed be driven by β-catenin. Since primary HGSOC tumors have great complexity in terms of heterogeneity, TOP-eGFP sorting-based studies in primary HGSOC tumors are a more ideal way to understand the regulation of the phenotype and platinum resistance, in the context of tumor heterogeneity by Wnt/β-catenin signaling. In addition, TOP-eGFP based cell sorting in primary HGSOC tumors could be an efficient means of identifying gene signatures that reliably stratify Wnthigh and Wntlow HGOSC tumors, thus enabling efficient Wnt/β-catenin-targeted therapy. Combination of cisplatin with CIC pathway-specific inhibitors in ovarian tumors could lead to personalized therapies targeting chemo-resistant subpopulations in ovarian tumors, thus forming an attractive approach for CIC-targeted therapy. Since there is extensive cross talk between the major signaling pathways regulating CIC phenotype the combination of CIC pathway-specific inhibitors with platinum-based regimens could greatly improve patient outcome in ovarian cancer.Intratumor heterogeneity in cancers is known to be associated with resistance to chemotherapy, and CICs are thought to play an important role in establishment and maintenance of this phenotype . Ovarian2) at 37°C. At 70-90% confluence, trypsin (0.25%)/EDTA solution was used to detach the cells from the culture plate for passaging and used for further experiments until passage 20. Cisplatin was purchased from Mount Sinai Hospital Pharmacy. 10mM stock solutions of iCG-001 (Selleckchem) were prepared in DMSO (Fisher Scientific) and stored at −20°C. The OV81.2 cell line was generated from re-engrafted tumors that were derived from the ascites of a patient diagnosed with HGSOC. After successful engraftment of tumor cells into immunodeficient mice, tumor fragments were isolated and propagated on tissue culture plates in-vitro. To confirm tumorgenicity and conserved patient histology of these primary HGSOC cells, 500,000 cells were injected subcutaneously into immunodeficient mice and tumors that formed were subjected to histopathological analysis to allow for comparison to primary patient tumor. The platinum-resistant derivative of OV81.2, namely, CP10, was generated by propagating OV81.2 in the presence of 2.5μM cisplatin for 10 passages.A2780 (cisplatin-sensitive) and CP70 (isogenic cisplatin-resistant to A2780) cells were obtained from Dr. Paul Modrich (Duke University). All cells used were cultivated in DMEM media supplemented with 10% FBS and 1% Penicillin-Streptomycin (recommended media). Cells were cultured in 10mm plates in a humidified atmosphere  according to manufacturer's instructions. For mRNA analysis, cDNA synthesis from 1μg of total RNA was done using the Transcriptor Universal cDNA Master kit (Roche). SYBR Green-based Real-time PCR was subsequently performed in triplicate using SYBR-Green master mix (Roche) on the Light Cycler 480 II Real-time PCR machine (Roche).For ALDH based sorting, ALDH assay was done using the ALDEFLUOR kit as per the protocol instructions (Stem cell Technologies) and ALDH positive and negative cells were FACS sorted using BD FACS Aria II. For ALDH activity assessment, the same kit was used and the data was acquired with Coulter Epics XL machine. For TOP-eGFP sorting, eGFP low and high populations were sorted from TOP-eGFP transduced stable cell lines using BD FACS Aria II.plus (Roche) method following manufacturer's instructions.Whole cell protein extracts were probed with antibodies against β-catenin (1:500)  and GAPDH (1:1000) (Santa Cruz) as previously described . MembranCells were plated in 12-well plate at 50,000 cells/well and treated the next day with the corresponding drugs. After 24hrs or 48hrs, cells were then incubated with 3- for 2hrs and absorbance was measured at 600nm. Isobologram analysis was done using GraphPad Prism software.Cell survival was assessed through seeding 1000 cells/well in a 6-well plate and treated with indicated doses of cisplatin once every 3 days. On day 7, cells were fixed in a 10% acetic acid/10% methanol (in diH2O) solution and stained with 1% crystal violet (in methanol) after 7 days of growth. Colonies were counted using Image J.Cells were plated 200,000 cells/well in a 6-well plate. The next day, cells were treated with drug and harvested 72 hours later. Annexin V/PI Staining was done using the FITC Annexin V Apoptosis Detection Kit II (BD Pharmigen). FACS data was acquired using the Coulter Epics XL machine.For tumor sphere formation assays, 1000 cells per ml were plated in 2ml in 6-well ultra-low attachment plate (Corning) in MammoCult medium (Stem cell Technologies) and after the indicated timepoint, 10×10 stitch imaging was done at 10x (100 random images acquired) using an Retiga Aqua Blue camera  connected to a Leica DMI6000 inverted microscope. Individual images were taken and then a composite image was generated using the scan slide function in Metamorph Imaging Software . Subsequent integrated analysis also used Metamorph software. For limiting dilution sphere assays, BD FACS Aria II sorter was used to sort cells directly into 96-well ultra-low attachment plates in 200μl mammocult media per well (Corning). After 7 days, the number of wells with tumor spheres was counted and the data was analyzed by Extreme Limited Dilution Analysis [ELDA] platform to determine the stem cell frequency.pTOP-Flash reporter construct stable cell lines were generated after lipofectamine 2000 [Life Technologies] based co-transfection of pTOP-flash construct with pBABE-puro plasmid [1:10 ratio] and puromycin-based selection. These were treated with iCG-001 and then cells were harvested after 8hrs and luciferase activities were analyzed using the Dual-Luciferase Reporter Assay System (Promega) with data normalization to the corresponding protein concentrationt-test (two-tailed) was used to compare two groups (P<0.05 was considered significant) for independent samples.Unless otherwise noted, data are presented as mean ± SD from three-independent experiments, and Student's


LiCl also suppressed β-catenin mRNA (p < 0.03), total β-catenin protein (p < 0.025) and the active form of β-catenin. LiCl-mediated inhibition of CRC cell proliferation was partially reversed by IWP-2, and Wnt9A antibody. Recombinant Wnt9A protein emulated LiCl effects by suppressing β-catenin protein (p < 0.001), inhibiting proliferation (p < 0.001) and increasing apoptosis (p < 0.03). This is the first study to demonstrate induction of a NCWP ligand, Wnt9A as part of a mechanism for LiCl-mediated suppression of CRC cell proliferation.Most studies of Wnt signaling in malignant tissues have focused on the canonical Wnt pathway (CWP) due to its role in stimulating cellular proliferation. The role of the non-canonical Wnt pathway (NCWP) in tissues with dysregulated Wnt signaling is not fully understood. Understanding NCWP’s role is important since these opposing pathways act in concert to maintain homeostasis in healthy tissues. Our preliminary studies demonstrated that LiCl inhibited proliferation of primary cells derived from colorectal cancer (CRC). Since LiCl stimulates cell proliferation in normal tissues and NCWP suppresses it, the present study was designed to investigate the impact of NCWP components in LiCl-mediated effects. LiCl-mediated inhibition of CRC cell proliferation ( Wnt signaling is an intricate ensemble of components of both canonical and non-canonical pathways involved with various processes including cellular proliferation, differentiation, motility and polarity . AlthougAn added layer of regulation is provided by the non-canonical pathway which also tempers canonical pathway activity . Thus, aMost studies of Wnt signaling in malignant tissues have focused on the canonical pathway due to its role in stimulating cellular proliferation. These studies have been instrumental in demonstrating that a prominent feature of Wnt signaling dysfunction in cancer cells involves the regulation of β-catenin. Due to mutations in either the gene(s) for β-catenin or the destruction complex, the protein is relieved from regulatory constraints and is able to stimulate target gene expression in an uncontrolled manner . It is iGiven the importance of the non-canonical pathway in normal tissue homeostasis, it is conceivable that components of the non-canonical pathway might also contribute to the abnormal proliferation of malignant tissues. Demonstration that non-canonical components are suppressed in some cancers ,9,10 is in vitro can be inhibited by the induction of Wnt9A that functions as a non-canonical ligand which results in suppression of β-catenin protein.Recent studies show that lithium chloride, a drug used for psychotropic disorders , suppresn = 4) resulted in a concentration-dependent suppression of cell proliferation by LiCl (p < 0.001) relative to media controls . In response to LiCl the total β-catenin levels were significantly decreased relative to media treated cells at 72 h . With respect to Wnt9A, LiCl increased expression by an average of 23-folds ; Wnt7A (p < 0.025); and the Wnt inhibitor SFRP4  that we observed in association with proliferation inhibition (ranging from 58% to 98%), coupled with the literature cited here, justified investigating the contribution of Wnt9A in these effects. In addition, the results obtained by: blocking ligand secretion; treatment with specific anti-Wnt9A antibody; and treatment with recombinant Wnt9A protein all point to a role for Wnt9A in the suppression of CRC cells we report herein. The suppression of β-catenin protein levels by Wnt9A which emulate LiCl effects further adds to this notion and indicates that the suppression of CRC cell proliferation is mediated in part through the activation of the non-canonical pathway that inhibits the canonical pathway. Nevertheless, while all these maneuvers produced results that were quantitatively significant and qualitatively consistent with a role for Wnt9A as a tumor suppressor, the magnitude of proliferation inhibition observed cannot be attributed to the effects of Wnt9A alone. Thus, other genes or factors must be involved.The substantial and consistent upregulation of NKD1 gene. In conjunction with Wnt9A, NKD1 was the only other gene that was significantly upregulated in all cell lines. NKD1 is a protein that functions as a negative feedback inhibitor of the Wnt pathway but has the capacity to modulate both canonical and non-canonical signaling pathways [Wnt9A to enhance the suppressive effects of Wnt9A observed with LiCl. Further studies are needed to determine this possibility.One such possibility is the pathways . It is, Dkk1, SFRP4, Wnt7A and Wnt8A. Dkk1 is a potent inhibitor of the canonical Wnt pathway as well as a target. The activation of the canonical pathway increases its expression which results in feedback inhibition of the pathway [SFRP4 which can bind to Wnt ligands and inhibit both pathways [Wnt7A and Wnt8A are considered non-canonical and canonical ligands, respectively [Wnt9A strongly suggests that further studies assessing the contribution of other non-canonical Wnt pathway components are justified.The contribution of Wnt pathway proteins that were significantly upregulated in response to LiCl in at least some of the 5 cell lines used in this study may also merit further investigation. Additional candidates based on our studies include:  pathway . This ispathways . The Wntectively . These cWnt9A has the potential to act as a tumor suppressor in cells that have sustained oncogenic mutations sufficient to hyperactivate β-catenin action. Studies that demonstrate decreased expression of the Wnt9A gene in different cancers including colorectal cancer are consistent with this possibility [Wnt9A expression is related to apoptosis and cell cycle arrest [Wnt9A leads to increased proliferation of breast cancer cells [Wnt9A gene sufficient to result in loss of function of the protein have been reported [The results presented herein suggest that the non-canonical Wnt ligand, sibility . Such a e arrest  as well er cells . In coloreported .Wnt9A was significantly increased by LiCl and that this ligand inhibited colorectal cancer cell proliferation in association with suppression of β-catenin expression  were established from resected tumors from patients with metastatic and/or recurrent disease. CRC were treated with LiCl , an activator of the canonical Wnt pathway (CWP) in the presence and absence of various Wnt pathway modulators including: IWP-2 (Sigma-Aldrich Corp.), a pan inhibitor of CWP and NCWP Wnt ligand secretion; conditioned media (CM) from LiCl ± IWP-2 treated cells; a specific antibody against Wnt ligand, Wnt9A ; and recombinant Wnt9A protein . Cell proliferation and apoptosis assays and RNA isolation for quantitative PCR gene expression array analysis were performed at 72 h. ELISA was performed on cell lysates at 24, 48 and 72 h.Short term, primary cell lines were established from resected colorectal tumor specimens, classified and characterized by the attending surgical pathologist on each case. The tumors were obtained under an institutional review board approved protocol, from five patients (age range = 40–64 years) who were undergoing treatment for colorectal adenocarcinoma. Short term, primary CRC cell lines  storage of the extra tissues not needed for clinical care; and (2) permission to conduct research on these tissues stipulating that all identifiers are stripped from the specimen and can only be matched to the patient and their clinical data through a code provided by the repository. The code is under the control of the principal investigator, Tan, Chair of the Department of Pathology and Laboratory Medicine, and not shared with any of the investigators who use the tissues in the repository.Tumor lines were passaged in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum  and 50 μg/mL Gentamicin . Tumor specimens received from surgery were dissected into 1 mm slices and subjected to a collagenase/DNase  digestion at 37 °C for 1–2 h. After digestion, specimens were strained, washed in HBSS (Sigma), and put into culture.2 First Strand cDNA Kit  according to the manufacturer’s instructions. Transcriptional gene expression was performed using the Human Wnt Signaling Pathway Plus RT2 Profiler PCR Array System , according to the manufacturer’s instructions. Real-time PCR was performed using the MyiQ Real-time PCR detection system . All transcriptional gene expression analyses were performed on the SABiosciences Corp. web portal using the RT2 Profiler PCR Array Data Analysis program (version 3.2). Transcriptional gene expression was defined as fold-change versus media controls.Transcriptional expression of genes was determined on the extracted RNA using 96-well real-time PCR arrays. cDNA was synthesized using the RT-Active-β-Catenin (anti-ABC), clone 8E7  [Total and active β-catenin levels were determined by ELISA by the Human Total β-Catenin DuoSet IC . To determine the active β-catenin levels, the capture antibody provided with the kit was replaced by AntiMA, USA) . Cells wCell proliferation was measured by a standard MTS Assay . A total of 5000 cells in logarithmic growth phase were inoculated into treatment wells in quadruplicate. Cells were subjected to various treatments (indicated above) and incubated for 72 h. Three hours before the end of the incubation period, MTS reagent was added to the plates and absorbance readings were performed on a multimode plate reader  at 490 nm.Cell apoptosis was determined using Caspase-3 Colorimetric Assay kit . Cells were subjected to various treatments for 72 h and lysed by the addition of the lysis buffer provided with the kit. The Caspase-3 assay was performed on cell lysates according to the manufacturer’s instructions using a multimode plate reader . The results are expressed as fold change relative to media controls. t-test and One-way analysis of variance (ANOVA) with post hoc pairwise multiple comparisons using the Student-Newman-Keuls method was performed to determine significance of differences between treatments. p-Values of less than 0.05 were considered significantly different.The data were expressed as means ± standard error of the means (S.E.M). Student’s Wnt9A as part of a mechanism for LiCl-mediated suppression of CRC proliferation. Inducing components of the non-canonical Wnt pathway may be therapeutically beneficial for attenuating uncontrolled proliferative canonical signaling in malignant tissues.This is the first study to demonstrate induction of a non-canonical ligand,


DPAGT1. Whereas these pathways have been studied independently, little is known about the dynamics of their interaction. Here we present the first numerical model of this network in MDCK cells. Since the network comprises a large number of molecules with varying cell context and time-dependent levels of expression, it can give rise to a wide range of plausible cellular states that are difficult to track. Using known kinetic parameters for individual reactions in the component pathways, we have developed a theoretical framework and gained new insights into cellular regulation of the network. Specifically, we developed a mathematical model to quantify the fold-change in concentration of any molecule included in the mathematical representation of the network in response to a simulated activation of the Wnt/ β-catenin pathway with Wnt3a under different conditions. We quantified the importance of protein N-glycosylation and synthesis of the DPAGT1 encoded enzyme, GPT, in determining the abundance of cytoplasmic β-catenin. We confirmed the role of axin in β-catenin degradation. Finally, our data suggest that cell-cell adhesion is insensitive to E-cadherin recycling in the cell. We validate the model by inhibiting β-catenin-mediated activation of DPAGT1 expression and predicting changes in cytoplasmic β-catenin concentration and stability of E-cadherin junctions in response to DPAGT1 inhibition. We show the impact of pathway dysregulation through measurements of cell migration in scratch-wound assays. Collectively, our results highlight the importance of numerical analyses of cellular networks dynamics to gain insights into physiological processes and potential design of therapeutic strategies to prevent epithelial cell invasion in cancer.The cellular network composed of the evolutionarily conserved metabolic pathways of protein N-glycosylation, Wnt/β-catenin signaling pathway, and E-cadherin-mediated cell-cell adhesion plays pivotal roles in determining the balance between cell proliferation and intercellular adhesion during development and in maintaining homeostasis in differentiated tissues. These pathways share a highly conserved regulatory molecule, β-catenin, which functions as both a structural component of E-cadherin junctions and as a co-transcriptional activator of the Wnt/β-catenin signaling pathway, whose target is the N-glycosylation-regulating gene,  DPAGT1, the first and key regulatory gene in the N-glycosylation pathway. Here, we describe the first mathematical model that accounts for the interactions among DPAGT1, E-cadherin, and Wnt/β-catenin signaling, revealing their key regulatory nodes. We use the model to make novel predictions, which we then validate experimentally by inhibiting β-catenin activation of DPAGT1 expression. We propose that this numerical model can be used to predict the network’s dynamics in cellular physiology and pathology.In epithelial tissues, protein N-glycosylation functions in a network with Wnt/β-catenin signaling and E-cadherin adhesion that maintains a balance between cell proliferation and intercellular adhesion. A key component of the network is β-catenin, a structural partner of E-cadherin junctions and transcriptional effector of Wnt/ β-catenin signaling that is also a transcriptional co-activator of  Certain cellular processes that are crucial for survival are highly conserved in evolution. These processes operate through a small set of proteins constituting a regulatory skeleton of cellular control . These rThe Wnt/β-catenin signaling pathway is highly conserved and acts as a regulator of development and cell fate –13. The DPAGT1 gene. Expression of glycosyltranferases that function late in the pathway has been shown to be coordinated with DPAGT1, emphasizing the importance of the initial steps in the LLO pathway [DPAGT1 is a target of the Wnt/β-catenin signaling pathway, thus linking this metabolic pathway with Wnt signaling [Protein N-glycosylation is a fundamental metabolic process in eukaryotes with up to 50% of proteins modified with N-glycans . The N-g pathway ,26. Indeignaling .DPAGT1 has been shown to affect the organization and assembly of AJs with the force sensing ability of AJs partly determined by the number and complexity of N-glycans modifying E-cadherin’s ECs [DPAGT1 itself acts as a common node in the network as it is the link between N-glycosylation and Wnt/β-catenin signaling. N-glycosylation of Wnt components impacts Wnt signaling as measured by the abundance of cytoplasmic β-catenin. The extent of N-glycosylation regulates the strength of the Wnt signal as both Wnt3a to LRP5/6 both are only efficiently secreted and positioned in the cell membrane, respectively, if properly N-glycosylated [N-glycosylation has been shown to affect the function of E-cadherin, the major epithelial cell-cell adhesion receptor –30. E-cain’s ECs ,36,37. Bin’s ECs ,39. DPAGosylated .et al., reaction kinetics were used to describe the concentration and effective half-life of β-catenin [et al. explored the impact of the existence of two β-catenin conformations in the cytoplasm, being the first group to model the dual role of β-catenin in the cell [et al. modeled the synergistic effect of two different Wnt inhibitors that act on transmembrane receptors components of the pathway, focusing on pathway activation [et al. addressed E-cadherin mediated adhesion and integrated β-catenin and E-cadherin binding dynamics into an agent-based model of epithelial sheets [DPAGT1. Agreement between simulated and experimental responses to activation of Wnt/β–catenin signaling with Wnt3a, provides confidence about the predictive capability of the RCN model.This schematic representing interactions among protein N-glycosylation, Wnt/β-catenin signaling and E-cadherin adhesion is insufficient, however, to predict cellular behavior. These relations are based on observations at equilibrium and under a limited set of conditions. Numerous biochemical reactions, frequently with opposing effects in cell behavior, cause this overall result. Furthermore, varying molecular concentrations in time can cause these pathways to affect each other in opposing ways. For this reason, a more thorough model is required, and with this added complexity comes the need for the use of mathematical modeling to quantify cellular responses in terms of molecular abundance. In a first reported model of the Wnt/β-catenin signaling pathway by Lee -catenin . Our RCN-catenin ,42. Sincthe cell . The modtivation . They wel sheets . The modl sheets . Our modDPAGT1 and N-glycosylation (reactions 11–19), and E-cadherin recycling and AJ formation (reactions 20–26).A reaction scheme was designed according to the current understanding of pathways comprising the RCN . The entet al. [et al. [Reactions 1–10 are simplified from the Lee et al.  and the  [et al. . Additio [et al. , the pro [et al. . Second, [et al. ,49,50. Treference condition. The complex formed by β-catenin and TCF was assumed to function as a transcription factor complex, driving the expression of DPAGT1 to generate the GPT enzyme. Transcriptional induction was modeled as a Hill-type activation. This has been demonstrated to be appropriate for this system [The reaction scheme was first represented mathematically as a set of ordinary differential equations (ODEs) with kinetic rates derived from literature ,42,49–52σ). This factor is a time-dependent continuous variable in the system that is calculated for each of the E-cadherin pools in the cell and represents the inverse degree of N-glycosylation of E-cadherin. The value is 0 when E-cadherin is the least adhesive (and most N-glycosylated), and it is 1 when the most adhesive (and least N-glycosylated). It is calculated for the E-cadherin pool in the ER upon synthesis of the E-cadherin/β-catenin complex as a function of GPT concentration and updated for other pools based on E-cadherin transport rates. This approach was chosen because micro-pipette aspiration studies have demonstrated that degree of N-glycosylation affects binding probability between adjacent cells [To describe the effect of N-glycosylation on AJ stability, the rates at which membrane E-cadherin/β-catenin complexes can assemble into or disassemble from AJs were made functions of an adhesivity factor (nt cells . A detaiσM), that is, inversely proportional to the degree of N-glycosylation of E-cadherin in the membrane. Similarly, the rate of AJ disassembly is made proportional to the negative of the adhesivity factor of E-cadherin in junctional complexes (σAJ). For more details see Appendix A. Expressions k24 represents the forward rate of homotypic E-cadherin binding, and k-24 is the backward rate.The rate of AJ assembly reaction 24 in 4 in σM, i.e. Wnt3a, APC, TCF, Axin/GSK-3β complex). The system of DAEs consists of 15 ODEs and 9 algebraic equations with 15 independent variables and 9 dependent variables, included in Appendix A. The dynamics are described by 35 parameters, with a complete list of parameter values along with their sources is included in To reduce the number of parameters, the ODEs were simplified into a system of differential algebraic equations (DAEs) based on two assumptions: rapid equilibrium approximation for reactions 1, 2, 6, 8, and 11  and consThe mathematical description of the system allowed us to test robustness to variations in parameters corresponding to different cellular conditions. Fold-change in the levels of β-catenin, rather than absolute abundance, has been shown to determine the extent of Wnt/β-catenin signaling activation and its effects on downstream targets ,53. For WNT0) and the Hill coefficient describing binding of the co-transcription factors to the DPAGT1 promoter. The former was excluded because it was used to define fold-change, the metric used for analysis, while the latter was excluded because the power term significantly complicates analysis. Additionally, Benary et al. showed the relative insensitivity of the system to the Hill coefficient [System sensitivity was tested by performing a local sensitivity analysis (LSA). This consisted of varying kinetic parameter values independently for Wnt “ON” and Wnt “OFF” states. The system of DAEs was solved at specific time points after activation with Wnt3a, and the corresponding fold-change values calculated. This approach identified the most revelant reaction in determining the fold-change response of each individual molecule in the RCN. Two parameters were excluded from LSA: total amount of Wnt3a (fficient . A more σ) to changes in each reaction in the RCN is shown in reference condition , the system reached steady-state at t = 35h. Analyses at 48h and 72h were included because the time it takes for the system to reach a steady-state was not measured for the different parameter values tested.The sensitivity of fold-change in cytoplasmic β-catenin, GPT, AJ, and the adhesivity factor  appeared to be slightly less sensitive than AJs to dysregulation of the RCN. Adhesivity appeared to be only highly sensitive to changes in TCF total abundance , the efficiency with which DPAGT1 is transcribed , and the rate of N-glycosylation , but not to changes in Wnt/β-catenin signaling.Adhesivity (σ) when disrupting β-catenin/TCF binding equilibrium  implies an evident variation in fold-change. A significant fold-change in both AJ and adhesivity was predicted only for perturbations around the reference value; for extreme perturbations, there was no change. This suggests that AJs and adhesivity are responsive to β-catenin translocation to the nucleus only within a certain parameter range. In contrast, fold-change in cytoplasmic β-catenin is sensitive to the shift in the direction of dissociation of β-catenin and TCF, but insensitive when equilibrium shifts towards formation of a stable complex.This reference and dysregulated conditions. This means that cells regulate the number of contacts much faster, and it is later that the stability of these contacts is determined with a period of hours between the two events, as suggested before [i.e. extent of dysregulation). By shifting the equilibrium between β-catenin and TCF towards unbinding, the drop in AJs was sharper and occurred first. With further dysregulation, however, recovery of junctions began with decreased N-glycosylation before restoring AJs. This suggests that in dysregulated epithelium, regulation of E-cadherin N-glycosylation can act against discohesion.AJ fold change reached a steady-state faster than adhesivity in both the d before . Despitedysregulated condition as a doubling of the equilibrium constant describing the binding between β-catenin and TCF  cells were kept under two conditions: a reference condition. The abundance of ABC increased on average by approximately 3 fold in the activated state compared to the constitutive state. An increase in the concentration of ABC was expected based both on the simulation predictions  and quantifying the difference of expression in the different conditions using immunoblot (IB). This provided an experimental measurement of the system’s response to activation with Wnt3a at the dictions  and repodysregulated condition and test the effectiveness of the RCN model, MDCK cells in constitutive and activated states were treated with ICG-001, a small molecule inhibitor of Wnt/β-catenin signaling. ICG-001 antagonizes β-catenin/TCF-mediated transcription by binding cyclic AMP response element binding protein (CBP), a transcriptional co-activator [To obtain the fold-change in a ctivator . Additioreference and dysregulated conditions for cells in both constitutive and activated states. This was done through immunoprecipitation (IP). Because α-catenin is recruited to mature junctional complexes, the stability of AJs was measured through the abundance of α-catenin in E-cadherin immunoprecipitates [constitutive state increased by approximately 1.5 fold when treated with ICG-001. In the activated state, the ratio of α-catenin to E-cadherin increased by approximately 2.7 fold. The treatment with ICG-001 caused intercellular adhesion to increase more drastically when Wnt/β-catenin signaling was activated  and peptide-H-glycosidase F (PNGaseF) and observing the mobility shift on immunoblots. EndoH specifically cleaves high mannose and hybrid N-glycans at the chitobiose core, while PNGaseF is an amidase that removes most N-glycans at the asparagine residues [To confirm the changes in adhesion with Wnt activation and network residues . Resulticonstitutive state  was used to monitor the optic flow within the cell sheet. In the th Wnt3a . In contth Wnt3a .The optical flow vectors also provide a way to quantify collectivity in the migration of the cellular monolayer. Collectivity is described by the correlation length, which is the average radius over which the correlation between lateral components of the PIV vectors are above a threshold value. More details on how this quantity is calculated can be found in the Methods section. The correlation lengths for the four experimental conditions are presented in movement angle) and mapping the occurrence of all possible movement angles for each instance of observation of the monolayer. A similar analysis has been performed to quantify collectivity in chemotactic experiments [Collective behavior of the cells was also analyzed by examining the directionality of optic flow in the entire MDCK monolayer in time. This is done by calculating the angle between the PIV vectors and the direction of wound closure . Specifically, we assessed how the predicted regulation of cell-cell adhesion through N-glycosylation affected cellular behavior. The observed dependence of AJ assembly on Wnt/β-catenin dysregulation is in accordance with the effect the overexpression of DPAGT1, increased N-glycosylation, and epithelial discohesion observed in MDCK cells and in oral squamous cell carcinoma [Because the RCN network plays a central role in development and its dysregulation is associated to the spread of cancer, we assessed the results of our theoretical analysis in the context of cell collectives and its implications to cell-cell adhesion and cell migration. Our experiments showed significant differences in motility and directionality when targeting a specific process in the network  to account for the effect of N-glycosylation of E-cadherin on adhesion at the molecular level. To date, this has only been addressed experimentally with single molecule force spectroscopy studies with the shortcoming that E-cadherin molecules were isolated from the rest of the junctional complex and cellular context [Beyond describing the change in AJ abundance, we defined the adhesivity factor , suggesting that DPAGT1/N-glycosylation functioned in the β-catenin/CBP branch of the Wnt/ β-catenin pathway. Given that HNSCC cells exhibit aberrantly upregulated β-catenin signaling and DPAPGT1 expression concomitant with hyper-glycosylated E-cadherin and greatly diminished cell-cell adhesion, the effects of ICG-001 were more pronounced in these cells compared to normal MDCK cells, which maintain coordinate regulation of cell proliferation and adhesion, and do not rely much on the β-catenin/CBP branch of Wnt/ β-catenin signaling. Indeed, MDCK cells require >2.5 fold greater concentrations of ICG-001 to bring about growth arrest. Thus, with the RCN model we can easily simulate the response of healthy and diseased cells to specific treatments. In summary, our studies provide an analytical tool that describes the dynamic response of single cells based on internal regulation and external stimuli. It can be used as a framework to choose therapeutics based on predicted effectiveness of promotion or inhibition of specific processes or integrated into agent-based models to account for the complex dynamics that arise from the intersection of three essential homeostatic pathways during collective processes such as morphogenesis and cancer.The agreement between protein measurements and cell behavior show the power of the RCN model to design new experimental approaches and predict outcomes. We note that for N-glycosylation the model was fit mostly using results from CK cells . The modki·X·Y—k-i·(X/Y) where X and Y denote the free concentrations of the binding partners, (X/Y) the concentration of the complex, and ki and k-i the association and dissociation rates respectively. Syntheses of proteins are described by constant rates (νi). Phosphorylation and dephosphorylation processes are described by linear rate equations (ki·X).In the system of ODEs, binding and dissociation processes are described by the rate equations: σ). This time varying factor is introduced as four variables, representing adhesivity of E-cadherin in each of the four pools included in the reaction scheme: endoplasmic reticulum (ER), membrane (M), endocytic recycling compartment (ERC), and adherens junctions (AJ) (fgain) and the adhesivity of the incoming and receiving E-cadherin, σsource and σdestination respectively. This change is described by To model the effect of the extent of E-cadherin N-glycosylation on homotypic E-cadherin interactions, an adhesivity factor was introduced (ons (AJ) . The chaσER is dependent on the concentration of GPT (X14) at the time of synthesis. The rate at which N-glycosylated E-cadherin is synthesized in the ER is calculated assuming Michaelis-Menten kinetics of GPT. σER over time:Because E-cadherin in the ER pool is synthesized and not transported from a source pool, Vmax represents the maximum rate of N-glycosylation of E-cadherin by GPT in the ER. Gmax represents the maximum possible concentration of N-glycosylated E-cadherin in the ER; this parameter was introduced to normalize the adhesivity factor (σ) to 1. KM represents the GPT concentration at which enzymatic activity is half-maximal. Derivation of this equation and its parameters’ values can be found in Appendices A and B.k24) on σM is described as a linear relation because at the large time scale explored and with multiple junctional complexes considered, homotypic E-cadherin bonds are persistent and do not vary in strength based on intercellular forces [k-24) on σAJ. A detailed explanation of the selection process of parameter values describing E-cadherin recycling rates and the slopes of the dependence of k24 on σM and k-24 on σAJ can be found in Appendix B  calculated for variation in the ith parameter over j values. F is the factor by which the physiological parameter value (j = p) has been multiplied to get the jth parameter value. To reach a single value of sensitivity of a variable to a single parameter, the values of Sij are averaged over all j. Steady-state concentrations for both Wnt “OFF” and “ON” conditions were using a numerical solver in Mathematica 10.2 .35 kinetic parameters were used to describe the 26 processes represented in 4cells/cm2 in DMEM media (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. To determine the effect of activating Wnt/β-catenin signaling, cells were serum starved (1% serum) for 24h then grown in the presence of 50% conditioned medium isolated from either L-mouse fibroblasts (controlled conditioned media) or L-mouse fibroblasts stably expressing Wnt3a cDNA (Wnt3a conditioned media) (ATCC). To determine the effect of a perturbation in the RCN, cells were either treated with 25 μM ICG-001 in 0.05% DMSO  or only DMSO as a control. When cells reached 30% confluency (3 days), cells were processed for preparation of total cell lysates (TCLs).For protein quantification, MDCK cells  were plated at 3x104cells/cm2 in a 24-well plate. Cells were kept in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were serum starved (1% serum) for 24h then grown in the presence of 50% conditioned medium (controlled conditioned media or Wnt3a conditioned media) (ATCC). To determine the effect of a perturbation in the RCN, cells were either treated with 10 μM ICG-001 in 0.1% DMSO  or only DMSO as a control. When cells became confluent (3 days), a scratch-wound assay was performed.To record collective migration dynamics, MDCK cells (II-G) with GFP conjugated E-cadherin  were plated at 6x10Aliquots of TCLs containing 300μg of protein were used for each immunoprecipitation reaction. First, aliquots were precleared with protein A/G PLUS-agarose beads  and a mouse IgG2a antibody  for 30 min at 4°C. Next, 2μg of mouse anti-E-cadherin antibody  was used for immunoprecipitation (2h at 4°C) followed by adsorption to protein A/G PLUS-agarose beads (1h at 4°C). For the isotype control, a mouse IgG2a antibody (same used for preclearing) was used in place of the anti-E-cadherin antibody. Immunoprecipitates were recovered by centrifugation , washed thrice with 1X-PBS, and boiled twice in 25μL 2X-SDS sample buffer. Elutions for each sample were combined and saved for immunoblot.TCLs were fractionated on 4–20% gradient SDS-PAGE, transferred onto PVDF membranes and processed as described . The folTotal cell lysate were digested with 100 units of either peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH), purchased from New England Biolabs, for 1 h at 37°C and analyzed by immunoblot following fractionation on 7.5% SDS-PAGE.http://www.micro-manager.org) employs a 10X 0.3 NA objective lens to image multiple ~576x576 μm2 FOV.When cells were confluent, approximately 3 days after exposure to conditioned media and ICG-001, the cell monolayer was scratched with a 200μL pipette tip. Immediately following this, cells were washed with media with same contents of conditioned media and ICG-001 to clear debris before replenishing media. Cells are placed on a stage incubator and imaged every 30 minutes for 15 hours. Two different positions within the sheet are imaged per condition: Half of the wound with the wound splitting the fields of view (FOV) in half, and a confluent area 100 μm behind the wound edge. Bright field and fluorescent images are acquired with a DMI600B Microscope  and ImagEM EM-CCD Camera  using a Spinning Disk Confocal setup . Micro-Manager 1.4 Software : The leading front is defined as the average foremost detected fluorescence along the wound. Leading edge speed is calculated by looking at the time it takes for the average front to reach the end of the FOV.PIV analysis is performed on bright field images of the confluent area 100 μm behind the wound edge to determine. The displacement field (optic flow) was calculated using an ImageJ plugin . This PI<…> represents the average. u(r) represents the difference between the lateral component (i.e. perpendicular to the direction of scratch) of a particular optic flow vector and average lateral velocity of all vectors. r’ represents the reference vector used to evaluate the correlation function with respect to vectors (r) at a distance r. The value of the correlation function is averaged over each value of r over all vectors from a single PIV analysis performed on a pair of sequential images. The correlation length is defined as the distance at which the correlation function becomes negligible, in accordance with the method used by Das et al. [The correlation length is calculated from the correlation function defined by s et al. . The corS1 TextAppendix A contains a list of all variables used to describe the system mathematically, a description of the different types of equations used in the system of ODEs, and a description of how the system of ODEs was simplified to a system of differential algebraic equations (DAEs). Appendix B contains a description of how the different parameters used to describe reaction rates were calculated or estimated.(DOCX)Click here for additional data file.S1 FigReaction labels refer to numbering used in (TIF)Click here for additional data file.S2 FigRed signifies a relative sensitivity to a parameter (column) of fold change in a variable (row) which is half of the average sensitivity of the variable to all parameters; green signifies a relative sensitivity to a parameter which is half of the average to all parameters. Average sensitivities values in bottom row are the average relative sensitivity values of fold change in all variables to a single parameter. All fold change values are calculated based on concentrations at steady-state.(TIF)Click here for additional data file.S3 FigTotal cell lysates were fractionated on 4–20% gradient SDS-PAGE, transferred onto the PVDF membrane and incubated with anti-ABC antibody  (TOP) followed by anti-GAPDH  antibody (BOTTOM). Immunoblot was developed using the chemiluminescence method (Thermo Scientific).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 VideoLeading edge of MDCK cell  monolayer. Cells were imaged for 15h (30min in between frames).(AVI)Click here for additional data file.S2 VideoLeading edge of MDCK cell  monolayer. Cells were imaged for 15h (30min in between frames).(AVI)Click here for additional data file.S3 VideoLeading edge of MDCK cell  monolayer. Cells were imaged for 15h (30min in between frames).(AVI)Click here for additional data file.S4 VideoLeading edge of MDCK cell  monolayer. Cells were imaged for 15h (30min in between frames).(AVI)Click here for additional data file.


Oral squamous carcinoma is the sixth most common cancer worldwide, and one of the most common cancers in developing countries. Regional and distant metastases comprise the majority of cases at initial diagnosis and correlate with poor patient outcomes. Oral epithelia is one of many tissue types to exhibit a cadherin switch during tumor progression, in which endogenous cell adhesion proteins, such as E-cadherin, give way to those of mesenchymal origin. The mesenchymal cell adhesion protein N-cadherin is found at the invading front of oral squamous carcinomas and has been strongly correlated with poor patient prognosis. The goal of the present study was to elucidate the mechanism by which N-cadherin may increase extracellular matrix-associated proteolytic activity to facilitate invasiveness in oral tumor development. The overexpression of N-cadherin in two oral squamous carcinoma cell lines increased motility, invasive capacity and synthesis of matrix metalloproteinase-9 (MMP-9) in a manner that was independent of E-cadherin downregulation. The use of EN and NE chimeric cadherin molecules with reciprocally substituted cytoplasmic domains revealed that optimal induction of MMP-9 synthesis required the cytoplasmic region, but not the extracellular region, of N-cadherin. Utilizing an N-cadherin mutant with impaired p120 binding ability, we found that such mutation resulted in a 4-fold decrease in motility compared to wild-type N-cadherin, but did not affect either MMP-9 expression or motility-normalized invasion. Overexpression of wild-type N-cadherin produced a 27-fold increase in the transcriptional activity of β-catenin, concomitant with increases in MMP-9 transcription. These results suggest that N-cadherin may promote motility and invasiveness through distinct mechanisms, and that β-catenin may be an integral mediator of N-cadherin-dependent invasive signaling in oral epithelia. Oral cancer is the sixth most common cancer worldwide, and is responsible for 128,000 deaths each year . In the Tumor metastasis is facilitated by a highly coordinated tandem of increased migratory ability coupled with increased proteolytic activity towards extracellular matrix components. The acquisition of such characteristics is one of the hallmark features of a cellular dedifferentiation program termed epithelial-to-mesenchymal transition (EMT) . An earlde novo expression of the mesenchymal adhesion protein N-cadherin  supplemented with penicillin, streptomycin and 10% fetal bovine serum (PAA). Murine fibroblast NIH3T3 cells (American Tissue Culture Collection) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum, penicillin and streptomycin.The oral squamous carcinoma cell lines Tu167  and SCC1cDNAs encoding full-length human N-cadherin , chimeriDetergent extraction of cell monolayers and SDS-PAGE was performed as described previously . The mou4 cells/chamber. Media conditioned by NIH3T3 cells  was used as a chemoattractant in the lower chamber. Membranes were collected after 24 (motility) or 48 (invasion) hours, and the upper surface of each insert membrane was scraped with a cotton swab to remove cells that had not traversed through to the other side. The remaining cells were stained with Diff-Quick (Dade) and counted. Quantitation was performed by counting cells in 9 random fields of view at 100× and expressing the average number of cells/field of view. All experiments were repeated in triplicate. The data are presented as the average of three independent experiments with the standard deviation of the average indicated. For presentation of the Invasion Indices, cell counts for invasion were normalized to the counts obtained for motility of each respective cell line, and expressed as a percentage compared to controls.Tu167 and SCC1 cells were plated in serum free media in 24-well Matrigel-coated or uncoated Boyden chambers  at a cell density of 7.5×10For zymographic analysis, confluent cell cultures in 6-well plates were incubated in serum-free MEM for 18 h and then in newly replaced collection media (serum-free MEM) for an additional 24 h. To normalize to cell number at time of media collection, cell monolayers were rinsed twice with phosphate-buffered saline and lysed with RIPA buffer. Volumes of conditioned MEM media proportional to the total recovered protein from each culture were analyzed for gelatinase activity according to the protocol of Leber and Balkwill . Gels weRenilla luciferase control vector (Promega). Lysates were collected for analysis 24 h after transfection. Firefly and Renilla luciferase activities were determined utilizing the Dual Glo Luciferase Assay Kit (Promega). The values reported were corrected for TCF/LEF-independent transcriptional activation and transfection efficiency. Data shown are the average of three independent experiments with standard deviations indicated.Cells were transfected with TOPFlash or FOPFlash firefly luciferase vectors, and a t method for relative gene expression utilizing GADPH values as an endogenous control.Total RNA was prepared using the High Pure RNA Isolation Kit (Roche). RNA (2 μg) was reverse-transcribed using the High Capacity cDNA Synthesis Kit (Invitrogen). Quantitative gene expression was performed for MMP-9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the TaqMan Gene Expression system with gene-specific primers and probes . PCR reactions were performed on a StepOne Plus Real-Time PCR System (Applied Biosystems) using TaqMan Universal PCR master mix according to standard manufacturer’s protocol. The data were then quantitated using the comparative COral squamous carcinoma cells are one of many tissue types that downregulate E-cadherin in response to the overexpression of mesenchymal cadherins ,34. The As shown in In prior studies utilizing a dexamethasone-inducible N-cadherin cDNA, SCC1 cells exhibited a scattered phenotype in response to increased expression of N-cadherin protein . SimilarGelatin zymography of conditioned serum-free media  was perfThe effect of altered N- and E-cadherin expression on both cell motility and invasion was examined by Transwell migration assay . RepreseAn oral squamous carcinoma cell model has previously been utilized to identify the fourth extracellular repeat domain EC4) of N-cadherin as the region responsible for conferring increased motility . In brea of N-cadThe EN chimera (EN), which lacked the N-cadherin extracellular domain, retained the ability to induce MMP-9 in both Tu167 and SCC1 cells . When thThe cytoplasmic region of N-cadherin interacts with two proteins that are known to play key roles in tumor progression: p120 catenin and β-catenin ,36. P120Consistent with previous studies ,29 cellsOne of the most well characterized interactions of the cytoplasmic portion of N-cadherin is with that of the protein β-catenin. β-catenin has been shown to function as a modulator of both cytoskeletal attachment and TCF/LEF-dependent transcription ,38. β-caet al, who demonstrated robust N-cadherin-dependent increases in MMP-9 only upon administration of fibroblast growth factor (The present study demonstrated that in oral squamous cells, the overexpression of N-cadherin, but not loss of E-cadherin, is sufficient to increase levels of MMP-9 transcript and protein levels  and 6C. h factor . In our h factor , furtherh factor  and 4.Because the extracellular domains of N-cadherin did not appear to play a role in MMP-9 induction , we turnA second binding partner of the cytoplasmic portion of N-cadherin is β-catenin, a growth-associated protein that is dually resident within the adherens junction and in the nucleus, where it functions as a coactivator of the Tcf/Lef family of transcription factors . In the The N-cadherin-dependent increase in β-catenin transcriptional activation is in discordance with previously proposed hypotheses that the sequestration of β-catenin within adherens junctions interfered with the ability of β-catenin to function as a transcriptional activator ,44. Suchet al (How then might N-cadherin overexpression positively regulate the nuclear activities of β-catenin? Recent research suggests that the initial association of β-catenin with cadherins may be vital to the ability of β-catenin to function as a transcriptional activator. In MDCK cells, the association of β-catenin with E-cadherin and the internalization of membrane-resident E-cadherin complexes were necessary to maintain β-catenin-mediated transcriptional activation of reporter genes . This meet al  have proet al . Recent et al .The present study demonstrates distinct roles of N-cadherin in promoting motility and invasion-related proteolysis. Although p120 is critical for increased motility, p120 does not appear to function in the induction of matrix metalloproteinases. Our data suggest that increased transcriptional activation of matrix metalloproteinases, and increased invasive capacity, was instead mediated by increased β-catenin transcriptional activity. Such regulation of β-catenin may have implications beyond increased matrix metalloproteinase activity, as β-catenin-dependent transcription is integral to a number of signaling pathways essential to tumor progression ,37. Furt


Axin, a β-catenin target gene, increased phosphorylation of Akt and GSK3, accumulation of β-catenin in the cytoplasm/nucleus in PC9M2 cells. Both knockdown of β-catenin and treatment with a β-catenin inhibitor at least partially restored gefitinib sensitivity to PC9M2 cells. Lung adenocarcinoma tissues derived from gefitinib-resistant patients displayed a tendency to accumulate β-catenin in the cytoplasm. We provide a rationale for combination therapy that includes targeting of the Akt-β-catenin pathway to improve the efficacy of EGFR-TKIs.There is a high death rate of lung cancer patients. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in some lung adenocarcinoma patients with EGFR mutations. However, a significant number of patients show primary and acquire resistance to EGFR-TKIs. Although the Akt kinase is commonly activated due to various resistance mechanisms, the key targets of Akt remain unclear. Here, we show that the Akt-β-catenin pathway may be a common resistance mechanism. We analyzed gene expression profiles of gefitinib-resistant PC9M2 cells that were derived from gefitinib-sensitive lung cancer PC9 cells and do not have known resistance mechanisms including EGFR mutation T790M. We found increased expression of  Lung cancer is the leading cause of cancer death worldwide2358911However, one serious problem that has arisen is that patients who initially had a good response to EGFR-TKIs ultimately experience a recurrence within only a few years. Several mechanisms of such acquired resistance to EGFR-TKIs have been reported. A secondary mutation of the EGFR, T790M, which impairs the binding between EGFR and gefitinib, was observed in many patients with acquired resistance13141618Activation of the Wnt/β-catenin pathway has been observed in a variety of cancers25262829In this study, we established gefitinib-resistant cell lines by long-term exposure of a gefitinib-sensitive cell line to gefitinib to explore novel mechanisms of resistance to EGFR-TKIs. Comparison of the gene expression pattern of gefitinib-resistant cell lines with that of the parental cell lines showed that Wnt/β-catenin signaling molecules were upregulated in the gefitinib-resistant cell lines. Furthermore, we demonstrated that sensitivity to gefitinib was associated with β-catenin activation. Our data suggest that combination therapy of β-catenin pathway-targeting drugs and EGFR-TKIs will be effective for adenocarcinoma patients with primary or acquired resistant to EGFR-TKIs. Our finding raises the intriguing possibility that activation of the Akt-β-catenin pathway might be a common mechanism for conferring resistance to cancer treatment, not only to EGF-TKIs, but also to other types of treatment, including chemotherapy and radiotherapy.Gefitinib-resistant PC9M2 cells were established by culturing gefitinib-sensitive PC9 cells, which harbor a 5 amino acid deletion in the EGFR tyrosine kinase domain7To explore the novel mechanism of acquired resistance to EGFR-TKIs in PC9M2 cells, we comprehensively analyzed gene expression profiles of PC9 and PC9M2 cells using DNA microarray analysis. Since we assumed that gefitinib or EGF-treatment may affect the gene expression levels during the time course, we analyzed the gene expression profiles over 24 h, one cell cycle of PC9 cells. Cells were treated with gefitinib and EGF for various periods of time, prior to mRNA extraction . The mRNAxin2, which is a typical target gene of β-catenin-induced transcriptionAxin2 were higher in PC9M2 cells than in PC9 cells  mice. We confirmed that gefitinib treatment significantly inhibited M2 cells . After 3M2 cells . Many cuM2 cells . These rFinally, we immunohistologically analyzed β-catenin localization in lung cancer tissues harboring EGFR TKI-sensitive mutations that were derived from patients who were subsequently treated with gefitinib. The clinicopathological characteristics of the patients are shown in the P = 0.049). These results suggest that localization of β-catenin was related with gefitinib sensitivity in lung adenocarcinoma patients.We then correlated the β-catenin staining pattern with the clinical effects of gefitinib-treatment, which were classified as follows: “partial response”; patients who responded partially (8 cases), “no response”; patients who did not respond significantly (21 cases)Axin2 gene transcription. We next demonstrated that downregulation of β-catenin using siRNA or an inhibitor of β-catenin activity, restored gefitinib sensitivity to PC9M2 cells, indicating that β-catenin activation conferred gefitinib resistance to the cells. Furthermore, we examined the activation of β-catenin in vivo using a mouse xenograft model. Plasma membrane localization of β-catenin was more prominent in the tumor derived from PC9 cells than in that derived from PC9M2 cells, whereas cytoplasmic and nuclear β-catenin localization were more evident in the tumor derived from PC9M2 cells than in that derived from PC9 cells. These observations indicate that β-catenin activation was enhanced in PC9M2 cells compared with PC9 cells in vivo.In this study, we established the gefitinib resistant cell line, PC9M2 that was derived from PC9 cells, which harbor an EGFR mutation. Neither the EGFR T790M mutation nor MET amplification was detected in the PC9M2 cells, suggesting the existence of other mechanisms of resistance to gefitinib in this gefitinib-resistant cell line. By comparison of the gene expression pattern of PC9 and PC9M2 cells, we found that Wnt/β-catenin-related genes were upregulated in PC9M2 cells. We confirmed that the β-catenin pathway is indeed activated in PC9M2 cells by determination of enhanced phosphorylation of Akt and GSK3α, cytoplasmic localization of β-catenin and activation of The major genetic abnormalities that are frequently observed in NSCLC are abnormalities in K-Ras and in receptor tyrosine kinases such as EGFR103839There are many reports that Wnt/β-catenin pathways play important roles in the maintenance of cancer stem cells2241Since the cellular localization of β-catenin should at least in part reflect the activity of the β-catenin pathway, we examined whether β-catenin localization in lung adenocarcinoma tissues with an EGFR mutation is associated with primary resistance to gefitinib. We observed that cytoplasmic localization of β-catenin is associated with lung adenocarcinoma tissues derived from primary gefitinib-resistant patients (no response cases) rather than with those from gefitinib-sensitive patients . We did not observe clear nuclear localization of β-catenin, which might be because β-catenin activation in the patient samples was not as strong as that in PC9M2 cell-derived tumors. However, it is possible that the β-catenin pathway is more highly activated in lung cancer tissues derived from patients who are resistant to EGFR-TKI treatment than in tissues derived from patients in whom the EGFR-TKI treatment is effective. It has been demonstrated that β-catenin activation is critical for tumorigenesis in an ErbB2/HER2-mediated mouse mammary tumor modelOur results raise the possibility that drugs targeted against the β-catenin pathway may be effective for patients whose lung cancer tissues display cytoplasmic localization of β-catenin. Since part of the adenocarcinoma tissues of gefitinib-sensitive patients display cytoplasmic localization of β-catenin, these patients may also be sensitive to drugs targeted against the β-catenin pathway. Obviously, it would be important to analyze β-catenin localization in lung cancer tissues derived from patients with acquired resistance to gefitinib. However, we did not have a large enough number of lung cancer tissues for such an analysis in the present study. Analysis of a large number of samples might be needed to validate the effect of drugs targeted against the β-catenin pathway on acquired resistance to gefitinib in patients.Recombinant human EGF was purchased from Merck Millipore . Gefitinib, MM-2206 and ICG-001 were purchased from Selleck chemicals .PC9 cells were maintained in RPMI1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin, at 37 °C and 5% CO2. To establish gefitinib resistant cell lines, parental PC9 cells were treated with gefitinib at concentrations of 20–100 nM for 14–28 days, 5–20 nM for 14–28 days and 100 nM for a range of exposure durations, yielding approximately 500 survived clones. We selected PC9-derived cells that showed a difference in gefitinib sensitivity but not in morphology or cell growth rate. Among the 500 clones that survived long-term exposure to gefitinib, we selected clones based on MTT assay. We treated the cells with 0, 0.1 and 1 μM of gefitinib, cultured them for 7 days and selected the clones that showed resistance to gefitinib, which resulted in a total of 179 clones (EGFR were 5′- TGT AAA ACG ACG GCC AGT CGA AAG CCA ACA AGG AAA TCC-3′ (forward) and 5′- CAG GAA ACA GCT ATG ACC ATT CCA ATG CCA TCC ACT TGA T-3′ (reverse). To quantitatively analyze the Axin2 mRNA expression level, quantitative real time PCR (qRT-PCR) was performed using a TaqMan probe as previously described in detailAxin2 (Hs00610344_m1) and for 18S ribosomal RNA (Hs99999901_s1) were purchased from Life technology .RNA isolation, cDNA preparation and sequencing were described previouslyEach cell line was starved for 48 h prior to the microarray experiments. Total RNA was then isolated from each line at 26 time points over a 24 h period. Hybridization was carried out on a 44K Agilent Whole Human Genome Oligo Microarray (G41112F) (Agilent Technologies). Microarrays were scanned using a dynamic autofocus microarray scanner  under default parameters (Green PMT was set to 100% and the scan resolution was set to 5 μm). Feature Extraction Software v9.1 (Agilent Technologies) was used to obtain a raw signal value and a quality flag . After aggregating all the scanned signals for the different time points, cell lines and treatments into a single data matrix, quantile normalization was applied to remove the between-experiment distributional biases due to assay artifacts by forcing the same percentile values on all of the processed data. To assign a unique probe ID to each gene that is inherently spanned by multiple probes, we selected a probe with the maximal variance across the overall data points. Outliers were further processed by comparing the distance between the data points and smoothing spline curve fitted under a degree of freedom and then replacing all of the data points lying above a threshold on the distance to the smoothed value. Finally, a gene filtering procedure based on a specifically customized statistical test (details are omitted) excluded genes that maintained expression levels for 24 h that were lower than a properly controlled threshold. These preprocessing procedures made up the eight time course microarray data from 17,654 genes.t-test was performed on the two sets of time-course measurements. For gene i and treatment j, the expression values of the parental PC9 and PC9M2 cells, YijtPC9 and YijtPC9M2, consisted of paired samples over the 26 time points . A one-sided test was used to assess the increased expression of gene i in the PC9M2 cells. We then controlled the false discovery rate of the multiple testing over 17,654 genes at q-values < 0.05G1, G2, G3 and G4 respectively) in the PC9M2 cells treated with control (j = 1), gefitinib (j = 2), EGF (j = 3) and both EGF and gefitinib (j = 4), respectively.To identify pathways involved in the maintenance of gefitinib resistance, we focused on genes whose expression was specifically elevated in the gefitinib resistant PC9M2 cells in spite of the presence or absence of gefitinib-treatment or EGF-treatment. For each treatment type, a paired two-sample ditPC9 = Yi1tPC9– Yi2tPC9 and ditPC9M2 = Yi1tPC9M2– Yi2tPC9M2. To assess the difference in expression of each gene in the drug response between the parental and PC9M2 cells, a two-sided paired t-test was carried out with the two differentiated time-course profiles, ditPC9 and ditPC9M2 . The produced p-values were converted to q-values. Controlling the false discovery rate at a q-value < 0.05, we obtained a set G5 of 6,421 genes that responded differently to gefitinib treatment across the cell types. The intersection of the five gene sets, G1, G2, G3, G4, G5, then resulted in 1,696 statistically significant genes that consisted of a set G.To further narrow-down the identified gene sets, we selected genes that were differentially regulated by gefitinib treatment in the parental PC9 and gefitinib resistant PC9M2 cells. Within each cell type, the difference in gene expression values was taken over the time points between the control and gefitinib-treated data sets, G. An overrepresentation test based on hypergeometric distributionbiological process’. This analysis detected strong enrichment of signals of the Wnt/β-catenin pathway, as the term GO:0016055 ‘Wnt receptor signaling pathway’ was ranked 9th (P = 0.003) in the list of 1,656 fourth-level ontology terms that were arranged according to increasing order of the p-values (www.ncbi.nlm.nih.gov/geo) under the accession number GSE34228.Finally, we investigated enrichment of Gene Ontology (GO) terms in the gene set p-values . Microar2, 100 mM NaCl, 1 mM DTT, 1 mM Na3VO4, protease inhibitor cocktail, phosphatase inhibitor-cocktail and 0.5% NP-40). Protein expression levels of EGFR, HER2 and HER3 were examined by immunoblotting using anti-EGFR (MBL), anti-HER2 , anti-HER3 (Millipore), anti-phospho-EGFR  and anti-phospho-HER2  antibodies respectively and anti-β-actin antibody (Millipore) as a loading control. Total and phosphorylated GSK3α were detected using anti-GSK3α  and anti-phospho GSK3α (Ser21)  antibodies at a 1:1000 dilution. Nuclear and cytosolic fractions were prepared as follows. Cells were treated with or without 1 μM gefitinib for 1 hour and were lysed in buffer A (10 mM HEPES-KOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail ). Cell lysate was centrifuged at 2,000 × g for 2 min at 4 °C. The pellets were resuspended in buffer C (50 mM HEPES-KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 2% glycerol, 1 mM DTT, 0.5 mM PMSF and protease inhibitor cocktail) and rotated at 4 °C for 30 min. Nuclear extracts were prepared by centrifugation at 15,000 × g for 15 min at 4 °C. The expression of β-catenin in nuclear or cytosolic fractions was detected by immunoblotting using an anti-β-catenin  and anti-HistonH3  antibodies and horseradish peroxidase-conjugated secondary antibodies specific to mouse or rabbit IgG (BD bioscience). Signals were detected by Immobilon (Merck Millipore).Cells were lysed in a lysis buffer (20 mM HEPES-NaOH (pH 7.5), 3 mM MgCl4 cells) were plated in a 12-Fwell plate and transfected with 50 nM siRNA using Lipofectamine® RNAiMAX Transfection Reagent according to the manufacturer’s protocol. At 72 h after transfection, cells were analyzed as described in the text.The β-catenin siRNA and control siRNA were purchased from Cell Signaling technology PC9 or PC9M2 cells .6 cells/mouse) (N = 6). From 6 days after injection, gefitinib (10 mg/kg/day) or vehicle was administrated orally everyday. After 2 ~ 3 weeks, the tumor was resected and immunohistochemically analyzed. Tumor volume was measured 3 times a week using the following formula: V = 1/2(L × W2), where L equals length and W equals width. Tumors were fixed in formalin, embedded in paraffin and then stained with hematoxylin-eosin (HE). Tissue sections were deparaffinized in xylene, rehydrated in graded ethanol and subjected to antigen retrieval. Immunohistochemistry was performed using the β-catenin antibody  at a 1:1000 dilution and signal was detected using EnVision (DAKO ChemMate). All experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee at Institute of Medical Science, University of Tokyo and Kanazawa University.PC9 cells or PC9M2 cells were injected subcutaneously into the flanks of 6-weeks-old female SCID mice  of tumor specimens with microwave antigen retrieval in 0.01 M citrate buffer (pH 6.0). Mouse monoclonal antibody against β-catenin at 1:200 dilution was used as the primary antibody and EnVision/HRP Polymer Reagent and DAB  Liquid (Dako) were used for detection.t-test according to the variance between the two samples. Statistically significant differences are indicated with an asterisk (P < 0.05), double asterisks (P < 0.01) or with triple asterisks (P < 0.001) in the figures. We used Barnard’s test for analysis of the contingency table47Data in the graphs are represented as means ± standard deviation (S.D.). Statistical significance was assessed using Student’s How to cite this article: Nakata, A. et al. Elevated β-catenin pathway as a novel target for patients with resistance to EGF receptor targeting drugs. Sci. Rep.5, 13076; doi: 10.1038/srep13076 (2015).


Mirror neurons fire during both action execution and observation of a similar action performed by another individual . HoweverOur analysis indicates that, at the level of single neurons, object/grip-specific decoders can be constructed, i.e. the type of the object/grip employed in either execution or observation can be decoded . However, only in 10% of the cases  made up the output vector Y. We assumed a linear relation between input and output as XW = Yand solved for the weights (the decoder parameters) using the pseudo-inverse solution W = X†Y. Then, given a 14-tuple vector representation, z, of a discharge, the predicted object id is given by W obtained in one condition was used to predict the object type in the other condition by using the data from that condition.Neuronal discharges during each condition were trimmed and represented as 14-bin histogram vectors. In the two neuron analysis, each neuron was reduced to a 7-bin histogram, and their concatenation results in a 14-tuple vector, to ensure similar decoder complexity (constant number of adjustable parameters). Thus, for each condition, ten 14-tuple neural firings  made up the rows of the input matrix


Its dysregulation is implicated as a driver of colorectal carcinogenesis and the epithelial-mesenchymal transition in other cancers. Nuclear β-catenin staining is a poor prognostic sign in synovial sarcoma, the most common soft-tissue sarcoma in adolescents and young adults. We show through genetic experiments in a mouse model that expression of a stabilized form of β-catenin greatly enhances synovial sarcomagenesis. Stabilization of β-catenin enables a stem-cell phenotype in synovial sarcoma cells, specifically blocking epithelial differentiation and driving invasion. β-catenin achieves its reprogramming in part by upregulating transcription of TCF/LEF target genes. Even though synovial sarcoma is primarily a mesenchymal neoplasm, its progression towards a more aggressive and invasive phenotype parallels the epithelial-mesenchymal transition observed in epithelial cancers, where β-catenin's transcriptional contribution includes blocking epithelial differentiation. SS18-SSX fusion oncogenes [SS18-SSX2 in specific cell lineages induces a completely penetrant and faithful recapitulation of SS in the mouse [Synovial sarcoma (SS) is the most common soft-tissue sarcoma diagnosed during adolescence and young adulthood . Clinicancogenes . Expresshe mouse . Both huβ-catenin functions as an epigenetic transcriptional cofactor in development and oncogenesis and drives the epithelial-mesenchymal transition in cancer . The AxiSS18-SSX2 in the mouse Myf5Cre lineage, suggesting near necessity of Wnt/β-catenin signaling to the transformation of that lineage [β-catenin stabilizing mutations and APC silencing mutations have been identified in SSs , 7. Nucl lineage . We inveex3f lCtnnb1 allele has loxP sites flanking the third exon [Δex3Ctnnb1, which expresses a shortened and stabilized β-catenin with normal trans-activation capabilities and a predilection for the nucleus [hSS2/wtRosa26; ex3fl/wtCtnnb1 embryos exposed to Cre-recombinase in culture demonstrated increased protein presence of the smaller, stabilized, Δex3fl form, despite equivalent transcription , necessitating another strategy for initiating synovial sarcomagenesis. First, we injected an adeno-associated virus (AdCre) into the bilateral hindlimbs of 7 four-week-old mice of each of 3 genotypes, hSS2/wtRosa26; wt/wtCtnnb1, wt/wtRosa26; ex3fl/wtCtnnb1, and hSS2/wtRosa26; ex3fl/wtCtnnb1. All mice carried a luciferase reporter allele  in the test dataset, normalized for the size of the gene set itself. Stringent q-values are less than 0.05.Microarray analysis of gene expression enabled comparison to extant data from prior models. Tumors from AdCre injected S2/CreER  mice, whA panel of mouse cancer models  with exphSS2/wtRosa26; ex3f l/wtCtnnb1 tumors could not be strictly attributed to the β-catenin stabilization alone, given the distinct tumor initiation method. Absence of control tumors developing within a year following AdCre mice limited opportunities to compare tumor phenotypes and transcriptomes. We next injected the protein TATCre [hSS2/wtRosa26; ex3fl/wtCtnnb1 mice  injections included 10Radiographs were obtained with a Kodak Carestream 4000ProFx , brightfield and fluorescent images with a Leica AF6000 dissecting microscope , and light photomicrographs with an Olympus BX43 microscope and DP26 camera .SS18-SSX2. The MoJoSS cell line, derived by dissociation and culture of a metastatic focus of monophasic SS in a lymph node, expresses SS18-SSX1, was maintained in DMEM with 20% FBS. The mouse cell line was derived from tumors that arose in a Myf5-Cre; hSS2Rosa26 mouse, was maintained in DMEM with 10% FBS. Validation of both developed cell lines also includes fusion gene expression and testing of dependence by siRNA, every 3 months in culture. SiRNA against mouse Ctnnb1  and human CTNNB1  were applied with Lipofectamine RNAiMax , using 3–4 μL of lipid reagent and 12–50 nM siRNA.The human SYO-1 cell line , receiveTotal RNA was isolated with RNeasy mini kit . Complementary DNAs were generated using Maxima first-strand (Fermentas Life Sciences). Quantitative PCR used the SYBR Green Master Mix  and an Applied Biosystems 7900HT. Primer sequences are in the Genome-wide expression profiling of AdCre tumors followed reverse transcription by One-Cycle Target Labeling with hybridization on the Mouse 430 2.0 Gene Chip . Microarray data were analyzed using DChip  and GeneFor transcriptome sequencing of TATCre tumors, RNA was prepared using the Illumina TruSeq RNA kit , checked with the Agilent Bioanalyzer RNA 6000 chip , captured using the RiboZero method (Illumina), and 50-cycle end-read sequenced on an Illumina HiSeq 2000.Reference fasta files were generated by combining the chromosome sequences from mm10 with all possible splice junction sequences, which were generated with USeq (v8.8.8) MakeTranscriptome using a radius of 46 and annotated with Ensembl transcripts (build 74) from the UCSC browser. Reads were aligned with Novoalign (v2.08.01), allowing up to 50 alignments per read. USeq's SamTranscriptomeParser selected the best alignment for each and converted the coordinates of reads aligning to splices back to genomic space. Differential gene expression was measured using USeq's DefinedRegionDifferentialSeq. Briefly, the number of reads aligned to each gene were calculated, then normalized in DESeq2 . R packaProtein was isolated using the M-PER reagent (Thermo Scientific). For cytoplasmic and nuclear fractions, tumors were homogenized to a single cell suspension. Cells were incubated 10 min in a mild lysis buffer and the cytoplasmic fraction was collected. The pellet was washed once in nuclear wash buffer, then purified over a 60% Percoll/sucrose gradient. The nuclear fraction was collected and lysed in nuclear lysis buffer on ice for 10 min. Both fractions were clarified at 14,000 rpm for 10 min before loading 25 μg/sample for gel electrophoresis and transfer to PVDF membrane. Western blots were probed with antibodies for β-catenin, c-MYC, Cyclin D1, H3, and GAPDH. Tissues were fixed in 4% paraformaldehyde overnight, decalcified (if including bone) in 14% ethylenediaminetetraacetic acid at pH 7.4 for two weeks at 4°C, and embedded in paraffin. Immunohistochemistry for mouse TLE1, BCL2, pan-cytokeratin, and epithelial membrane antigen were performed using IgG-horse radish peroxidase and hematoxylin counterstain.