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Many tight junction family genes also function elsewhere in the cell , and particular NEC cell lines may or may not have normal tight junctions .

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We ask however whether other genes selectively expressed in the NEC cell lines have additional epithelial-related functions , which would further test the inference that NEC gene expression provides a signature for epithelial character of tumor cells.Having defined an NEC cell line signature based on selective expression of a subset of tight junction and cadherin family genes , we used the pattern comparison tool of CellMiner to identify other genes selectively expressed ( or selectively not expressed ) by those NEC cell lines .

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We found 76 genes whose z-score correlations with respect to selective expression in the NEC cell lines was r > 0.75 .

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For each of those 76 genes , we assembled information about molecular interactions and functions from recent scientific literature .

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We found relevant information for 44 of the genes ( “ NEC-correlated epithelial genes ” ; Table 2 ) ; the remaining 32 had no published information linking them to epithelial-specific functions ( Table 3 ) , but these genes likely have functions in epithelial tumor cell lines that remain to be discovered .

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Genes that exhibited the strongest negative correlations with respect to selective expression in the NEC cell lines ( “ NEC-anti-correlated genes ” ) are listed in Table 4 .

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Those genes may have non epithelial or mesenchymal functions .

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Gene Expression Dichotomy between Epithelial-like and Mesenchymal-like Cell Lines .

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NEC-correlated epithelial genes from Table 2 and NEC-anti-correlated genes from Table 4 were combined in a clustered image map ( CIM ) of mRNA expression in the NCI-60 cell lines ( Figure 10 ) .

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As expected , they show a sharp dichotomy between epithelial-like and non epithelial cell lines with the NEC cell lines in a tight cluster ( upper red box ) .

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Interestingly , 8 of the 9 melanoma cell lines cluster together ( bottom red box ) , suggesting that gene expression patterns in these cell types differs from other non epithelial cell types .

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Particularly notable in these melanoma cell lines is that they tend to express ZEB2 selectively , but not ZEB1 ( arrows at bottom of the CIM ) .

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We then asked whether this gene expression dichotomy , which was based on NCI-60 data , would hold up in CCLE cell lines .

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We found that this is clearly true for CCLE cell lines derived from breast , colon , and ovary ( Figures 11 – 13 ) .

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Moreover , these mRNA expression CIMs allow us to estimate the fraction of the cell lines from each tissue type that have a non epithelial or mesenchymal gene expression pattern ( Table 5 ) .

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The values for the breast and colon lines were very close to those that were based on expression of tight-junction and adherens junction genes ( Figures 5 and 6 , Table 5 ) .

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The CCLE ovarian cancer cell lines had a relatively large percentage of non epithelial or mesenchymal-like cell lines ( 65 % , Figure 13 ) , perhaps due to the large incidence of ovarian tumors of mesothelial origin , which perhaps have a non epithelial gene expression profile ( the non epithelial cluster divides into two sub-clusters that perhaps distinguish between predominantly mesenchymal versus mesothelial character ) .

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The gene sets in Tables 2 and 4 thus distinguish epithelial-like from mesenchymal-like character in human tumor cell lines .

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The next question is whether those gene sets participate in a coherent functional network .

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In the current work we address that question for the epithelial-related genes in Table 2 , as well as interacting genes whose NEC correlation is significant , although not high enough to meet the criteria for inclusion in Table 2.In the following description of the molecular interactions of NEC-correlated genes , the first occurrence of a gene name in each section is shown in bold , along with the correlation value ( r ) for selective expression in the NEC lines , as given by CellMiner .

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Molecular Interactions and Signaling at Cell-cell Junctions of Epithelial-like Cancer Cells .

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Many of the most highly NEC-correlated genes were found to interact in a molecular interaction network related to cell-cell junctions : tight junctions , adherens junctions , and desmosomes ; these genes are colored red in the molecular interaction map ( MIM ) in Figure 14 ) .

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The relevant network interactions and functions of the NEC-correlated genes implicated in these functions are described below .

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At its first occurrence in each section , the name of each of those genes is in bold type along with its NEC expression correlation ( r ) .

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Interactions at Tight Junctions .

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The central components of tight junctions include members of the claudin family of genes , which encode tetra-spanning transmembrane proteins that associate laterally to form circumferential anastomosing bands near the apical region of epithelial cells .

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Their extracellular domains , which associate intercellularly in the space between adjacent cells , regulate ionic paracellular permeability between apical and basolateral regions of the extracellular space , and allow ion permeation with selectivity that differs among different claudins [ 13 ] [ 14 ] } [ 6 ] .

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The claudins whose expressions correlated most closely with the NEC cell line pattern were CLDN3 , 4 and 7 ( r = 0.76 , 0.80 and 0.93 , respectively ) ( Figure 4 ) .

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Note that the expression pattern for CLDN7 ( r = 0.93 ) is a nearly perfect match to the NEC pattern .

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Closely associated with the claudins in tight junctions is OCLN / occludin ( r = 0.58 ) , although its precise role in tight junctions is not clear .

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When epithelial cells migrate during wound healing , OCLN in complex with INADL ( r = 0.69 ) moves from cell-cell junctions to the leading edge of the migrating cells [ 15 ] .

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Also included in the tight junction structure are MARVELD3 ( r = 0.95 ) , a tetraspanning transmembrane protein [ 8] and MARVELD2 / tricellulin ( r = 0.77 ) , which is localized at 3-cell junctions in the epithelial monolayer [ 10 ] , [ 14 ] .

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Note that MARVELD3 , like CLDN7 , exhibited a nearly perfect match to the NEC pattern ( Figure 2 ) .

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Tight junction structures include members of the TJP / zona occludens family , of which only TJP3 / ZO-3 ( r = 0.87 ) correlated strongly with the NEC gene expression pattern ( Figure 4 ) .

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TJP proteins link tight junctions with the cortical actin cytoskeleton and are required for its structural integrity [ 16 ] – [ 18 ] .

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Possibly also involved is CGN / cingulin ( r = 0.80 ) , which can bind both TJP1 – 3 and actomyosin [ 19 ] , [ 20 ] ( Figure 14 ) .

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The TJP involvement may differ among cell types .

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We find TJP3 most prominently correlated with the expression of other NEC genes .

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In the CCLE colon cancer cell lines , however , TJP2 correlated in the same cluster with the NEC genes ( Figure 6 ) , and TJP1 appeared in the NEC-correlated gene cluster in the CCLE ovarian cancer cell lines .

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Thus , while TJP3 was selectively expressed in epithelial-like cancer cell lines , TJP1 and 2 , which are known also to participate in tight junction structures , may have more general functions in most of those cell lines.Directly associated with tight junctions are CRB3 / Crum3 ( r = 0.81 ) and INADL / Patj ( r = 0.69 ) ( Figure 14 ) , which bind to each other and are part of a complex that maintains apical / basolateral polarity of epithelial cells [ 21 ] , [ 22 ] .

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This complex is down-regulated upon epithelial-mesenchymal transition [ 23 ] .

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CRB3 also binds LLGL2 ( r = 0.80 ) , which participates in the complex that maintains apical / basolateral polarity .

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LLGL2 was able to reverse an epithelial-mesenchymal transition [ 24 ] .

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Tight junctions are also affected by the trans-membrane glycoproteins EPCAM / TACSTD1 / TROP1 ( r = 0.84 ) and TACSTD2 / TROP2 ( r = 0.64 ) , both of which bind CLDN7 ( Figure 14 ) .

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In the absence of EPCAM , CLDN7 protein ( but not its mRNA ) is depleted and the barrier function of tight junctions is impaired [ 25 ] .

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Unlike EPCAM , which is expressed in various epithelia , TACSTD2 is expressed in stratified epithelia , but not in colonic or other simple epithelia [ 26 ] .

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EPCAM binds CLDN7 tightly and inhibits its degradation , but does not localize at tight junctions .

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Instead it localizes at lateral cell-cell junctions , where it sequesters CLDN7 in regions distinct from tight junctions [ 27 ] .

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Although localized similarly to adherens junctions , EPCAM does not bind CDH1 / E-cadherin .

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These actions of EPCAM impair tight junctions and promote metastasis [ 25 ] , [ 27 ] .

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This unusual circumstance of an NEC-correlated gene associated with perturbation of epithelial cell-cell junction structures suggests a possible abnormality of epithelial cancer cell lines in culture , which however remains to be tested in normal epithelial cells .

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One possibility is that EPCAM is associated with epithelial cell proliferation during wound healing , and that epithelial cancer cell lines in culture proliferate as in wound healing , thus explaining the highly NEC-correlated EPCAM expression .

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Consistent with this possibility , EPCAM induces transcription of cyclin D1 ; in the absence of EPCAM , cyclin D1 , phosphorylated-Rb and cell cycle progression are suppressed [ 28 ] .

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The highly NEC-correlated gene LNX1 / PDZRN2 / MPDZ ( r = 0.78 ) codes for a PDZ domain-containing E3 ubiquitin ligase that targets CLDN3 , as well as serine / threonine kinase PBK and other proteins [ 29 ] .

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LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced cell sensitivity to doxorubicin [ 29 ] .

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LNX1 may have a role in tight junction organization or turnover through its association with CLDN1 , CLDN3 and TJP1 [ 30 ] , [ 31 ] .

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The manner in which the actions of LNX1 are functionally integrated however remain to be elucidated .

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Interactions at Adherens Junctions .

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Closely associated with tight junctions are adherens junctions , of which CDH1 / E-cadherin ( r = 0.77 ) is the major structural component .

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Additional components of adherens junctions are CAMSAP3 ( r = 0.76 ) and PLEKHA7 ( r = 0.52 ) , which bind to each other in a complex that could bring together several components : CAMSAP3 binds the minus ends of microtubules , and PLEKHA7 binds CTNND1 / p120-catenin ( r = 0.48 ) , which in turn binds CDH1 / E-cadherin [ 20 ] , [ 32 ] , [ 33 ] ( Figure 14 ) .

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The complex formed by these bindings links adherens junctions to microtubules .

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PLEKHA7 also binds CGNL1 / paracingulin , which binds CGN ( possibly indirectly ) [ 20 ] , thereby potentially linking between adherens junctions and tight junctions .

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Such linkage however would not be effective in the NEC cell lines , because these plastic-grown cells did not express CGNL1 .

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Adherens junctions are disassembled when CDH1 / E-cadherin is taken into endosomes , an action that is promoted by ARF6 and inhibited by ADAP1 / CENTA1 ( r = 0.82 ) .

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Thus ADAP1 , whose expression is highly NEC-correlated , maintains adherens junctions and preserves epithelial character [ 34 ] , [ 35 ] ( Figure 14 ) .

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Interactions at Desmosomes .

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Desmosomes confer strong cell-cell adhesion in association with adherens junctions in epithelial cells and provide linkage to the cytoskeleton , particularly keratin intermediate filaments .

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The interactions of the desmosomal proteins are shown in Figure 14 .

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Desmocollins , such as DSC2 ( r = 0.60 ) , and desmogleins , such as DSG3 ( r = 0.38 ) , are desmosomal cadherins that form calcium-dependent cell-cell junctions similar to those of the adherens junctions of CDH1 / E-cadherin .

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Plakophilins , such as PKP3 / plakophilin ( r = 0.71 ) , and JUP / plakoglobin ( r = 0.70 ) , constitute the outer dense plaque that connects to the desmosomal cadherins and to the cytoskeletal linker protein DSP / desmoplakin ( r = 0.62 ) on the cytoplasmic side of the plasma membrane ( Figure 14 ) .

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PKP3 is transcriptionally repressed by ZEB1 , an NEC-negatively correlated gene ( r = – 0.58 ) , thereby loosening epithelial cell-cell adhesion and promoting cell invasion and metastasis [ 36 ] .

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In stratified epithelia , desmoplakin links desmosomes to intermediate filaments .

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Another desmosome component , PPL / periplakin ( r = 0.76 ) , binds EVPL / envoplakin ( r = 0.49 ) and the two bind intermediate filaments [ 37 ] .

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PPL associates with ANXA9 / annexinA9 ( r = 0.81 ) ; the two proteins , whose expression is highly correlated with the NEC genes , co-localize at cell-cell junctions of epithelial cells [ 38 ] .

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ANXA9 has been reported to be up-regulated in prostate and colon cancers [ 39 ] , but details of its function have not been elucidated .

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The PPL-EVPL dimer associates with desmosomes via the N-terminal region of PPL [ 40 ] .

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Thus the mRNA expressions of 5 of the 7 above-mentioned epithelial desmosomal proteins correlated strongly ( r > 0.60 ) with the NEC gene expression pattern ( Figure 14 ) , while the remaining 2 correlated at lower but yet significant levels .

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Figure 14 shows how desmosomal proteins contribute to epithelial-specific interactions , several of which are mediated by JUP / plakaglobin .

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JUP / plakoglobin ( r = 0.70 ) is a structural component of both desmosomes and adherens junctions , where it binds the cytoplasmic tail of CDH1 / E-cadherin [ 36 ] , [ 41 ] ( Figure 14 ) .

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Plakoglobin helps maintain epithelial character and low proliferation rate .

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It binds to and inhibits the pro-mesenchymal actions of nucleophosmin ( Figure 14 ) , and may thereby convert mesenchymal cells to an epithelial-like state with low proliferation rate and reduced invasiveness [ 42 ] .

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In conjunction with TCF / LEF , plakoglobin functions as a transcription factor , which can act as a switch to induce expression of DSC2 and repress DSC3 [ 43 ] .

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Although DSC2 / desmocollin ( r = 0.60 ) was highly correlated with the NEC gene expression pattern , DSC3 lacked significant correlation .

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Thus Plakoglobin may signal LEF1 to transcriptionally activate the desmosomal cadherin DSC2 ( Figure 14 ) , which functions in epithelial cells as opposed to mesenchymal cells .

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Plakoglobin also binds and stabilizes NME1 / NM23-H1 via CTNNA1 / alpha-catenin , which would tend to inhibit metastasis [ 44 ] , [ 45 ] ( Figure 14 ) .

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Plakoglobin binds DSG3 / desmoglein ( r = 0.38 ) , which prevents entry of plakoglobin into the nucleus [ 46 ] ( Figure 14 ) .

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Thus JUP / plakaglobin is central to several epithelial-specific functions shown in the molecular interaction map in Figure 14 .

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DSP / desmoplakin ( r = 0.62 ) binds the epithelia-specific keratin intermediate filaments KRT8 ( r = 0.63 ) , KRT19 ( r = 0.63 ) and KRT18 ( r = 0.59 ) .

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In intestinal epithelium , these keratins function to maintain proper architecture of microvilli even without linkage to desmosomes [ 47 ] .

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However in stratified epithelia , keratins appear to be required to maintain desmosomes .

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During epithelial-mesenchymal transition , disassembly of desmosomes preceded loss of E-cadherin [ 48 ] .

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KRT8 / 18 reduced the sensitivity of human carcinoma cell lines to cisplatin , and depletion of KRT8 / 18 increased cisplatin-induced apoptosis [ 49 ] .

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The expression of KRT8 was closely correlated with that of BTG4 ( r = 0.54 relative to NEC ; r = 0.92 relative to KRT8 ) , which has anti-proliferative properties [ 50 ] .

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Indirect Interactions with Epithelial Cell Surface Complexes .

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Several NEC-correlated genes interact indirectly with tight or adherens junctions , regulate their functions , and link them to other cell structures ( Figure 14 ) .

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The largest number of interactions impacting cell surface complexes emerge from transcription factors GRHL1 and GRHL2 ( r = 0.83 and 0.89 ) , suggesting key rolls in the functions in the of epithelial cell-cell junctions ( Figure 14 ) .

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In particular , GRHL1 and / or 2 up-regulate the transcription of CLDN4 and CDH1 , as well as RAB25 ( r = 0.94 ) , whose expression is almost perfectly correlated with the NEC genes .

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RAB25 also enhances the expression of CLDN4 , induces its localization in tight junctions , and activates the transcription of TACSTD2 ( r = 0.62 ) , a binder of CLDN7 at tight junctions [ 51 ] ( Figure 14 ) .

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TACSTD2 is expressed in stratified epithelia , but not in colonic or other simple epithelia [ 26 ] ) .

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GRHL2 promotes transcription of ERBB3 ( r = 0.54 ) , which forms an oncogenic heterodimer with ERBB2 ( r = 0.37 ) in breast cancer cells [ 52 ] .

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