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Data Analysis . | PMC2825202 | -1 | [] |
Cycle threshold ( C ) is defined as the PCR cycle number at which the fluorescence generated from amplification of the target gene within a sample increases to a threshold value of 10 times the standard deviation of the base line emission and is inversely proportionate to the starting amount of the target cDNA . | PMC2825202 | -1 | [] |
QBasePlus was used for calculation of PDCD4 expression relative to each of the EC genes . | PMC2825202 | 16,786 | [
0
] |
It applies Ξ Ξ C method was used where Ξ Ξ Ct = ( C target gene , test sample - C endogenous control , test sample ) - ( C target gene , calibrator sample - C endogenous control , calibrator sample ) . | PMC2825202 | -1 | [] |
Relative quantities were corrected for efficiency of amplification and fold change in gene expression between groups was calculated as E - Ξ Ξ Ct Β± s.e.m . | PMC2825202 | -1 | [] |
Where more than one endogenous control are used , fold change estimates were calculated using the geometric mean of EC quantities relative to the calibrator sample which could be the minimum , maximum or a named sample or an average.Stability of the EC genes expression was evaluated with two freely available statistical models , geNorm and NormFinder . | PMC2825202 | -1 | [] |
It is further validated with qBasePlus . | PMC2825202 | 16,786 | [
5
] |
Statistical analysis was carried out with Minitab 15 ( Minitab Ltd , Coventry , UK ) . | PMC2825202 | -1 | [] |
Anderson-Darling normality test was applied and parametric tests were used where appropriate . | PMC2825202 | -1 | [] |
The equivalence test was used to assess the equivalently of expression of the candidate genes between tumour and normal tissues . | PMC2825202 | -1 | [] |
One-way ANOVA , two-sample t-test , Levene 's test and Spearman and Pearson correlations were used to determine association and comparisons between groups . | PMC2825202 | -1 | [] |
P values < 0.05 were considered statistically significant . | PMC2825202 | -1 | [] |
Authors ' contributions . | PMC2825202 | -1 | [] |
EAHK performed the experiments , was responsible for data analyses and drafted the manuscript . | PMC2825202 | -1 | [] |
KHC contributed to sample preparation from clinical samples and collation of clinicopathological data . | PMC2825202 | -1 | [] |
NM conceived , designed and supervised experimental work and manuscript editing . | PMC2825202 | -1 | [] |
JN contributed to statistical analysis of clinical data and drafting of the manuscript . | PMC2825202 | -1 | [] |
MJK contributed throughout the experiment , critically reviewed the manuscript and participated clinically . | PMC2825202 | -1 | [] |
All authors read and approved the final manuscript . | PMC2825202 | -1 | [] |
Supplementary Material . | PMC2825202 | -1 | [] |
Additional file 1 . | PMC2825202 | -1 | [] |
Table 1 Supplementary data . | PMC2825202 | -1 | [] |
Post hoc testing of individual levels of EC gene expression.Click here for file | PMC2825202 | -1 | [] |
Gene Expression Correlations in Human Cancer Cell Lines Define Molecular Interaction Networks for Epithelial Phenotype . | PMC4062414 | -1 | [] |
Using gene expression data to enhance our knowledge of control networks relevant to cancer biology and therapy is a challenging but urgent task . | PMC4062414 | -1 | [] |
Based on the premise that genes that are expressed together in a variety of cell types are likely to functions together , we derived mutually correlated genes that function together in various processes in epithelial-like tumor cells . | PMC4062414 | -1 | [] |
Expression-correlated genes were derived from data for the NCI-60 human tumor cell lines , as well as data from the Broad Institute 's CCLE cell lines . | PMC4062414 | -1 | [] |
NCI-60 cell lines that selectively expressed a mutually correlated subset of tight junction genes served as a signature for epithelial-like cancer cells . | PMC4062414 | -1 | [] |
Those signature cell lines served as a seed to derive other correlated genes , many of which had various other epithelial-related functions . | PMC4062414 | -1 | [] |
Literature survey yielded molecular interaction and function information about those genes , from which molecular interaction maps were assembled . | PMC4062414 | -1 | [] |
Many of the genes had epithelial functions unrelated to tight junctions , demonstrating that new function categories were elicited . | PMC4062414 | -1 | [] |
The most highly correlated genes were implicated in the following epithelial functions : interactions at tight junctions ( CLDN7 , CLDN4 , CLDN3 , MARVELD3 , MARVELD2 , TJP3 , CGN , CRB3 , LLGL2 , EPCAM , LNX1 ) ; interactions at adherens junctions ( CDH1 , ADAP1 , CAMSAP3 ) ; interactions at desmosomes ( PPL , PKP3 , JUP ) ; transcription regulation of cell-cell junction complexes ( GRHL1 and 2 ) ; epithelial RNA splicing regulators ( ESRP1 and 2 ) ; epithelial vesicle traffic ( RAB25 , EPN3 , GRHL2 , EHF , ADAP1 , MYO5B ) ; epithelial Ca ( +2 ) signaling ( ATP2C2 , S100A14 , BSPRY ) ; terminal differentiation of epithelial cells ( OVOL1 and 2 , ST14 , PRSS8 , SPINT1 and 2 ) ; maintenance of apico-basal polarity ( RAB25 , LLGL2 , EPN3 ) . | PMC4062414 | 1,172 | [
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The findings provide a foundation for future studies to elucidate the functions of regulatory networks specific to epithelial-like cancer cells and to probe for anti-cancer drug targets . | PMC4062414 | -1 | [] |
Introduction . | PMC4062414 | -1 | [] |
Progress in cancer biology and therapy depends in large part on comprehending the molecular interactions that govern key regulatory networks . | PMC4062414 | -1 | [] |
The vast amount of data on gene expression in cancer cells should assist in reaching that goal , but effectively utilizing that information remains challenging . | PMC4062414 | -1 | [] |
Most malignant solid tumors derive from epithelial tissues and retain epithelial characteristics to a variable degree that correlates inversely with malignant virulence . | PMC4062414 | -1 | [] |
We aimed to utilize gene expression data for cell lines derived from various human tumors to elucidate molecular interaction networks controlling functions key to epithelial cell types , leading eventually to deeper understanding of the factors that govern transitions to mesenchymal character , a change that is thought to be central to acquisition of the ability of cancer cells to invade tissue and form distant metastases . | PMC4062414 | -1 | [] |
The current work focuses on genes that are expressed selectively in epithelial cells , while a subsequent communication will focus on transitions between epithelial and mesenchymal cell states.Epithelia are arguably the best defined as well as the embryonically earliest multicellular phenotype . | PMC4062414 | -1 | [] |
A prominent characteristic essential to epithelia is tight junctions , which help to hold adjacent cells together and regulate transport of molecules through the paracellular space between adjacent cells [ 1 ] . | PMC4062414 | -1 | [] |
Expression of a subset of genes that are associated with tight junctions may therefore serve as an indicator of epithelial character . | PMC4062414 | -1 | [] |
This would be in accord with the general principle that genes that are expressed together in a variety of circumstances or cell types are likely to function together.The relative expression levels of over 23,000 genes in the National Cancer Institute β s 60 human tumor cell lines ( NCI-60 ) have been assembled into a freely and readily accessible database [ 2 ] . | PMC4062414 | -1 | [] |
In a previous study , we showed that a set of mutually expression-correlated genes over the NCI-60 cell lines could be assembled into networks that control cell migration [ 3 ] . | PMC4062414 | -1 | [] |
We now show that a subset of the NCI-60 cell lines that are selective in expression of certain tight junction-associated genes serve as a signature for epithelial character of tumor cells , and that genes positively correlated with that signature can be assembled into networks involved in the control of epithelial functions . | PMC4062414 | -1 | [] |
We show that the expression patterns in the NCI-60 human cancer cell lines correlates well with expression in the CCLE / Broad cell lines.Although gene expression at the mRNA level is not the sole determinant of corresponding protein expression ( for which we do not yet have adequate data ) , the function correlations are striking.The current work combines gene expression correlations with molecular interaction information directly from current scientific literature to assemble molecular interaction networks specific to epithelial-like cells . | PMC4062414 | -1 | [] |
In addition to the bioinformatics analysis , an integral part of this study includes a comprehensive review of molecular interactions of genes ( and gene products ) having epithelial-related functions in human cancer cell lines . | PMC4062414 | -1 | [] |
Methods . | PMC4062414 | -1 | [] |
Gene expression profiles and correlations for NCI-60 human tumor cell lines were obtained using the β Gene transcript level z score β web-based tool provided by CellMiner ( http://discover.nci.nih.gov/cellminer/ ) . | PMC4062414 | -1 | [] |
This tool provides relative quantitation for the cell lines from five microarray platforms [ 2 ] . | PMC4062414 | -1 | [] |
CellMiner provided z-score correlations ( r ) of the expression of a given gene with respect to selectivity for the NCI-60 epithelial consensus ( NEC ) cell lines ( see below and Table 1 ) . | PMC4062414 | -1 | [] |
Of the 22,379 genes for which there were validated data for the NEC genes in the CellMiner database , the fraction of genes having z-score ( r ) values greater than 0.90 , 0.80 , 0.70 , 0.60 , 0.50 , 0.40 were respectively 0.0005 , 0.0023 , 0.0056 , 0.013 , 0.027 , 0.059.Gene expression data ( CCLE_Expression_Entrez_2012-09-29.gct ) for human tumor cell lines of the Cancer Cell Line Encyclopedia ( CCLE ) of the Broad Institute of MIT and Harvard were downloaded from ( http://www.broadinstitute.org/ccle/data/browseData?conversationPropagation=begin ) . | PMC4062414 | -1 | [] |
The downloaded file was pre-processed using a combination of UNIX commands and R programs , e.g ., to remove entries for which the gene name was missing . | PMC4062414 | -1 | [] |
The expression values for each gene were converted to a z-score across all samples in the dataset ( i.e ., mean zero and unit standard deviation ) , using the R program scale . | PMC4062414 | -1 | [] |
The resulting matrix of gene expression profiles was saved as an R object . | PMC4062414 | -1 | [] |
An in-house R package was used to compile and normalize the data from individual samples into a coherent dataset for each cancer type . | PMC4062414 | -1 | [] |
The expression values for each gene were converted to a z-score across all samples in the dataset ( i.e ., mean zero and unit standard deviation ) , using the R program scale ( ) . | PMC4062414 | -1 | [] |
The resulting matrix of gene expression profiles was saved to hard drive as an R object.Clustered image maps for gene expression and correlations were generated using an in-house R package.Information on molecular interactions and functions was assembled from recent literature in PubMed . | PMC4062414 | -1 | [] |
The number of cited references was limited by citing recent publications that contain citations to earlier literature . | PMC4062414 | -1 | [] |
The molecular interaction maps ( MIMs ) were prepared using the notation described by Kohn et al [ 4 ] and at http://discover.nci.nih.gov/mim/ . | PMC4062414 | -1 | [] |
The MIM symbols used in the current work are defined in Figure 1 . | PMC4062414 | -1 | [] |
The MIMs in the current work were constructed using the PathVisio-MIM software [ 5 ] . | PMC4062414 | -1 | [] |
Results and Discussion . | PMC4062414 | -1 | [] |
Expression Pattern of a Subset of Tight-junction and Adherens-junction Genes in the NCI-60 Cell Lines Defines a Candidate Epithelial Signature . | PMC4062414 | -1 | [] |
Tight junctions are bands of specific structural proteins that seal cell-cell junctions and regulate passage of small ions or molecules through the intercellular space ; they are an essential characteristic of epithelial cell types [ 6 ] , [ 7 ] . | PMC4062414 | -1 | [] |
The structural core of tight junctions is generally composed of one or more proteins from each of the following genes or gene families : TJP1-3 , claudins ( CLDN1-27 ) , OCLN / occludin , MARVELD3 , and MARVELD2 / tricellulin [ 8] . | PMC4062414 | -1 | [] |
We asked whether a subset of those tight-junction-family genes would exhibit an expression pattern of mutually correlated genes within the NCI-60 panel of human tumor cell lines . | PMC4062414 | -1 | [] |
That pattern of selective expression could be a signature for epithelial character of human tumor cell lines in culture.Using the CellMiner NCI-60 analysis tools [ 2 ] , we found that 7 members of the tight-junction-family genes formed a consensus pattern of mutually expression-correlated genes in 11 of the 60 NCI-60 cell lines . | PMC4062414 | -1 | [] |
Figure 2 shows how closely those gene expression profiles resemble each other.Structurally associated with tight junctions are adherens junctions whose central component is the epithelial marker , CDH1 / E-cadherin . | PMC4062414 | -1 | [] |
Because of that functional relationship and the close similarity of its NCI-60 expression profile with that of the tight-junction genes displayed in Figure 2 , we included CDH1 in an epithelial consensus signature ( Figure 3 , Table 1 ) . | PMC4062414 | -1 | [] |
The high mutual expression correlation of the genes listed in Table 1 is seen in Figure 4 . | PMC4062414 | -1 | [] |
Selective expression of those mutually correlated genes therefore was selected as a possible signature for epithelial character of human tumor cell lines in culture . | PMC4062414 | -1 | [] |
We refer to those genes and the NCI-60 cell lines selectively expressing them as the β NCI-60 epithelial consensus ( NEC ) β signature . | PMC4062414 | -1 | [] |
Although selective expression of NEC genes may be indicative of epithelial character , it may or may not indicate the presence of normal tight and adherens junction structures . | PMC4062414 | -1 | [] |
Figure 3 shows that the NCI-60 gene expression profile for CDH1 / E-cadherin ( and thus of the NEC genes in general ) is nearly a mirror image of that of the mesenchymal marker gene , VIM / vimentin , suggesting that mesenchymal genes have selectively low expression in the NEC cell lines . | PMC4062414 | -1 | [] |
The genes whose expression was highly selective in the NEC cell lines may be epithelial-like , and the genes whose expression was selectively low in the NEC cell lines may be mesenchymal or , more generally , non-epithelial.The expression correlations of the NEC genes and cell lines in the context of all NCI-60 cell lines and all tight junction and cadherin gene family members is shown as a clustered image map ( CIM ) in Figure 4 . | PMC4062414 | -1 | [] |
We see that the NEC genes cluster is a subset of the tight junction and adherens junction gene families.In addition to the NEC genes listed in Table 1 , Figure 4 suggests that CDH3 ( P-cadherin ; correlation of gene expression with that of the NEC genes , r = 0.55 ) could be included in the cluster . | PMC4062414 | -1 | [] |
CDH3 will be seen to co-cluster with the bone fide NEC genes in other data sets ; therefore we consider CDH3 to be an ex-officio member of the NEC group.Many of the gene family members included in the CIM do not have epithelial-related functions or are expressed at plasma membrane regions other than tight junctions . | PMC4062414 | -1 | [] |
CDH2 / N-cadherin , for example , forms adherens junctions in mesenchymal cells , which do not have cell-cell junctions of the type that is unique to epithelial cells [ 9 ] . | PMC4062414 | -1 | [] |
Claudins ( CLDNs ) differ in their abilities to seal cell-cell junctions , and some of them form anion - or cation-specific channels in the narrow space between adjacent epithelial cells [ 10 ] . | PMC4062414 | -1 | [] |
Expression of Tight-junction and Cadherin Family Genes in the CCLE Human Tumor Cell Lines Derived from Epithelial Tissues . | PMC4062414 | -1 | [] |
Since the NCI-60 contain a limited number of cell lines per tissue type , we asked whether the NEC subset of tight-junction and cadherin family would also be evident in the data for the much larger number of human tumor cell lines of the Broad Institute β s Cancer Cell Line Encyclopedia ( CCLE ) [ 11 ] . | PMC4062414 | -1 | [] |
Using the same set of genes that were used in the CIM of gene expression in the NCI-60 cell lines ( Figure 4 ) , we prepared CIMs of mRNA expression in CCLE breast and colon cell lines ( Figures 5 and 6 , respectively ) . | PMC4062414 | -1 | [] |
These CIMs show that the NEC genes listed in Table 1 ( except for OCLN , for which we found no data in CCLE ) cluster together both in the CCLE breast and colon lines ( Figures 5 and 6 , red box ) , as they did in the CIM for the NCI-60 ( Figure 4 ) . | PMC4062414 | -1 | [] |
In addition to the 7 NEC genes , those clusters included CDH3 / P-cadherin in the breast lines and TJP2 / ZO-2 in the colon lines . | PMC4062414 | -1 | [] |
Figure 5 suggests that 16/58 ( 28 % ) of the CCLE breast cancer cell lines are non epithelial . | PMC4062414 | -1 | [] |
For the CCLE colon cancer cell lines ( Figures 6 and 7 ) , the corresponding fraction is 7/61 ( 11 % ) . | PMC4062414 | -1 | [] |
The expression correlations between gene pairs are shown in CIMs for CCLE breast and colon lines in Figures 8 and 9 , respectively . | PMC4062414 | -1 | [] |
For both the breast and the colon lines , the 7 NEC genes appear in a tight mutually correlated cluster ( Figures 8 and 9 , red box ) . | PMC4062414 | -1 | [] |
These findings support the idea that the NEC genes serve as a signature for epithelial character in human tumor cell lines derived from epithelial tissues , and show minor variations of the composition of the NEC gene cluster in cancer cell lines from various tissues . | PMC4062414 | -1 | [] |
In addition , both the breast and colon CIMs show a tight inversely correlated cluster ( Figures 8 and 9 , blue box ) , consisting of CDH2 , CDH4 , CDH6 , CDH11 , CDH13 , and CLDN11 , except that the breast cluster also contains MARVELD1 . | PMC4062414 | -1 | [] |
These genes thus tend to be down-regulated in the CCLE breast and colon cancer cell lines , and may function primarily in non epithelial or mesenchymal cell types ( which is well known for CDH2 / N-cadherin ) . | PMC4062414 | -1 | [] |
Thus the co-expressed NEC subset of tight-junction and cadherin family genes ( Table 1 ) is also evident in the CCLE human tumor cell lines derived from epithelial tissues.Most of the colon cell lines uniquely also express CDH17 ; selective expression of CDH17 is seen in 35 of the 54 ( 65 % ) epithelial-like CCLE colon lines ( Figure 6 ) . | PMC4062414 | -1 | [] |
Among those 35 cell lines , 21 ( 60 % ) also express CLDN2 ; and among those 21 lines , 6 ( 29 % ) additionally express CLDN15 ( Figure 6 ) . | PMC4062414 | -1 | [] |
Selective expression of CDH17 stands out as being specific to a large fraction of colon cell lines ; this was also apparent in the NCI-60 gene expression profile where CDH17 was selectively expressed in 4 of the 7 colon cell lines ( Figure 7 ) . | PMC4062414 | -1 | [] |
Thus colon cancer cells , and perhaps also colon cancers , may be stratified on the basis of expression of these genes . | PMC4062414 | -1 | [] |
Expression of CDH17 in colon cancer cell lines , as well as colon cancer tissues , was previously reported by [ 12 ] . | PMC4062414 | -1 | [] |
High expression of CDH17 was associated with reduced survival of colorectal cancer patients . | PMC4062414 | -1 | [] |
CDH17 was found associated with beta1 integrin and other factors suggesting effects on cell adhesion and extracellular matrix interactions . | PMC4062414 | -1 | [] |
In the CCLE ovarian cancer cell lines , approximately half of the lines showed high expression of CLDN16 , although the distinction between epithelial-like and mesenchymal-like cell lines was not clear . | PMC4062414 | -1 | [] |
The Cell Lines of the NCI-60 Epithelial Consensus ( NEC ) Serve as a Seed to Discover other Genes that are Selectively Expressed by these Cell Lines and that have Epithelial-specific Functions . | PMC4062414 | -1 | [] |