AFF3 mediates the oncogenic effects of β-catenin in ACC. The Wnt response element site located at nucleotide position −1408 of the AFF3 transcriptional start sites (TSS) mediates the regulation by the Wnt/β-catenin signaling pathway. AFF3 silencing decreases cell proliferation and increases apoptosis in the ACC cell line H295R. AFF3 is located in nuclear speckles, which play an important role in RNA splicing. AFF3 overexpression in adrenocortical cells interferes with the organization and/or biogenesis of these nuclear speckles and alters the distribution of CDK9 and cyclin T1 such that they accumulate at the sites of AFF3/speckles. We demonstrate that AFF3 is a new target of Wnt/β-catenin pathway involved in ACC, acting on transcription and RNA splicing.Adrenocortical cancer (ACC) is a very aggressive tumor, and genomics studies demonstrate that the most frequent alterations of driver genes in these cancers activate the Wnt/β-catenin signaling pathway. However, the adrenal-specific targets of oncogenic β-catenin-mediating tumorigenesis have not being established. A combined transcriptomic analysis from two series of human tumors and the human ACC cell line H295R harboring a spontaneous β-catenin activating mutation was done to identify the Wnt/β-catenin targets. Seven genes were consistently identified in the three studies. Among these genes, we found that  In progressive patients, medical therapy is of very limited efficacy. Several studies show the importance of the Wnt/β-catenin signaling pathway in the development and maintenance of numerous organs, and that alterations of the Wnt/β-catenin signaling are involved in a wide range of human diseases, and especially malignancies.6Adrenocortical cancer (ACC) is a very aggressive tumor with a 5-year survival rate below 35% in most series.Ctnnb1 in mice results in adrenal gland aplasia.7 Gene alterations leading to a constitutive activation of this pathway are the most frequent events in ACC.8 Constitutive activation of the Wnt/β-catenin pathway in the adrenal cortex of transgenic mice leads to the development of adrenocortical tumors with malignant characteristics.10 Inhibition of the Wnt/β-catenin pathway in the adrenocortical cell line H295R by PKF115-584 or a shRNA against β-catenin messenger RNA (mRNA) increases apoptosis12 and is associated with a complete absence of tumor growth in a xenograft model.12The Wnt/β-catenin pathway is required for normal adrenal (NA) gland formation, and adrenocortical cell-specific knockout of AXIN2, have been shown to be upregulated in ACC,13 but there has been no comprehensive and integrative study to identify all specific Wnt/β-catenin targets in ACC. A better understanding of the biological processes affected by Wnt/β-catenin pathway alterations in ACC may allow the identification of therapeutic targets and thereby contribute to the development of new therapeutic strategies. The aim of this study was to use a combination of transcriptome analysis and cellular experiments to identify the targets of the Wnt/β-catenin pathway involved in adrenocortical tumorigenesis.These observations implicate alterations of Wnt/β-catenin in ACC pathogenesis. Few Wnt/β-catenin target genes, including the canonical target We performed a combined transcriptomic analysis on cohorts of ACC and on human cell models to identify alterations of gene expression due to aberrant Wnt/β-catenin pathway activation.10 were analyzed for genes whose expression is correlated with the common Wnt/β-catenin pathway target gene, AXIN2  and 1 mRNA was negatively correlated   .CTNNB1 (β-catenin) gene mutation affecting the GSK3β phosphorylation site (S45P) and leading to constitutive transcriptional activity of β-catenin-lymphoid enhancer binding factor (LEF)/T-cell factor (TCF). Whole-transcript gene expression was analyzed in three stable clones of H295R cells expressing a doxycyclin-inducible small hairpin RNA (shRNA)-targeting CTNNB1 mRNA (shβ).12 We established a list of genes showing similar expression profiles in the three clones after CTNNB1 silencing. A control clone was used to eliminate from this list those genes whose expression was sensitive to doxycyclin treatment. We thereby identified 44 genes significantly downregulated  and 29 genes significantly upregulated  by specific CTNNB1 inactivation ; AF4/FMR2 family, member 3 (AFF3); family with sequence similarity 19 (chemokine (C–C motif)-like), member A4 (FAM19A4); isthmin 1; angiogenesis inhibitor (ISM1), acid phosphatase-like 2 (ACPL2); and naked cuticle homolog 1 (Drosophila) (NKD1)  (NKD1) . Two of CTNNB1 mRNA, and in established tumors with CTNNB1 inactivation from a subcutaneous xenograft model  silencing in adrenocortical cells H295R leads to increased apoptosis , AFF3 silencing had the largest effect on apoptosis  than in ACC without such nuclear staining (βcat−) and in NA in both cohorts  or after dichotomization between ACC with low and high AFF3 expression .14 To assess the transcriptional activity of AFF3 in human adrenal cells, we mapped transcriptome sequencing reads with mRNA (RNA-seq) from H295R cells and we examined the regions surrounding the two putative TSSs  were inserted in tandem into the reporter plasmid, pGL3Tk, where luciferase expression is driven by the minimal Tk promoter (2xWT or 2xMut). In transfected cells the transcriptional activity of the reporter construct (2xWT) was higher than that of the mutated construct (2xMut) (P<0.01). The transcriptional activity of the 2xWT was lower  in H295R cells expressing a shRNA-β-catenin (shβ with dox). Doxycyclin treatment did not affect activity of the 2xMut construct with mutated LEF/TCF sites. These results are consistent with the profiles of the canonical reporter constructs TOP/FOP-Flash  and recruitment of the activator β-catenin. The ive TSSs . There wtive WRE . In H295to AXIN2 . LEF1 siA levels . Two copOP-Flash .AFF3 gene, and differ owing to the alternative splicing of exon 4 without frameshift. PCR analysis of cDNA using oligonucleotides corresponding to parts of exon 3 and exon 5 showed that the mRNA for isoform 1 (v1), without exon 4 , was the most abundant of the two mRNAs in adrenocortical cells . Five days following shRNA-AFF3 induction by doxycyclin (dox) treatment, AFF3 mRNA and protein levels were significantly decreased (AFF3 expression in control lines (Ctr) (AFF3 silencing by shRNA (+dox) increased the apoptosis rate and increased caspase activity (AFF3 silencing increased the apoptotic effect of staurosporin (staurosporin had no significant effect on controls). The effect of AFF3 silencing on H295R cell proliferation was measured by MTT -2,5-diphenyltetrazolium bromide) and colony-formation assays: silencing significantly decreased proliferation  or the control line.Adrenocortical cell lines carrying constructs encoding a doxycylin-inducible shRNA targeting ecreased . In contes (Ctr) . AFF3 siactivity , a resulactivity . Likewis15 which serve as a reservoir of factors participating in mRNA splicing. AFF (AF4/FMR2) proteins have been also described in the super elongation complex (SEC), which contains the positive transcription elongation factor b (P-TEFb) and other elongation factors,16 and positively regulates transcriptional elongation by the RNA polymerase II. In adrenocortical cells, AFF3 colocalized with the SC35 protein (a nuclear speckle marker) (AFF (AF4/FMR2) proteins have been found in nuclear speckles, marker) , line 1. marker) , line 2  marker) . To dete marker) , top. Th marker) , middle  marker) , bottom.17 We and others have demonstrated the importance of the Wnt/β-catenin signaling pathway activation in adrenal cortex tumorigenesis.23 Using combined transcriptomic analysis we have identified new target genes of the Wnt/β-catenin signaling pathway. We show that one of these genes, AFF3, mediates some of the effects of Wnt/β-catenin signaling pathway activation in ACC . We also report that the WRE site located at nucleotide position –1408 of the AFF3 TSS, mediates the regulation by the Wnt/β-catenin signaling pathway.In many cancers, the Wnt/β-catenin signaling pathway has an important role in regulating cell growth, motility and differentiation.n in ACC . We haveAFF1 , AFF2 , AFF3 and AFF4 . AFF2 is silenced in cases of Fragile XE (FRAXE) intellectual disability. Proteins encoded by AFF1, AFF3 and AFF4 are produced as fusions with that encoded by MLL in acute lymphoblastic leukemia patients.26 In the mouse, Aff3 shows strong regional expression in the developing brain, somites and limb buds.29 This expression pattern is consistent with mesomelic dysplasia and central nervous system abnormalities observed in a female infant with a microdeletion on chromosome 2, only affecting the AFF3 gene.28 A recent study identified a CGG repeat expansion, associated with intellectual disability, in the promoter of AFF3 at an autosomal folate-sensitive fragile site named FRA2A.14 This polymorphic repeat is hypermethylated in FRA2A, leading to silencing of AFF3 in the nervous system.14Aff3 is expressed in the mouse adrenal cortex during embryonic development (E14.5) and thus may be implicated in adrenal development  family of genes includes four members: elopment . AFF3 ha pathway . Moreovepression , which i15 Several lines of evidence point to speckles being storage/assembly/modification compartments that supply splicing factors to active transcription sites.39 CDK9 and cyclin T1, two major components of P-TEFb involved in transcriptional elongation via phosphorylation of the RNA polymerase II, are diffusely distributed throughout the nucleoplasm with an overlap with nuclear speckles.39 We report that in H295R adrenocortical cells, AFF3 interacts with CDK9 and cyclin T1, and its overproduction alters the nuclear distribution of these two proteins such that they accumulate at the sites of AFF3/speckles. AFF3 and P-TEFb form the SEC-like 3 recently described in HEK293 cells.16 The SEC consists of the RNA polymerase II elongation factors eleven-nineteen Lys-rich leukemia (ELL) proteins, EAF1/2 (ELL-associated factor) proteins, P-TEFb and several frequent myeloid/lymphoid or mixed-lineage leukemia translocation partners, such as AF9, ENL, AFF1, AFF4 and AFF3. It is one of the most active P-TEFb-containing complexes and is required for rapid induction of transcription by paused RNA polymerase II. The significant overlap of genes downregulated by AFF3 silencing with genes controlled by CDK9 (determined by inhibition of CDK9 by Flavopiridol40) suggests that AFF3 modulates P-TEFb activity in adrenocortical cells , B-cell lymphoma 2 (BCL2) and cyclin-dependent kinase 6 (CDK6).43 It would therefore be worth pursuing the development of inhibitors that more directly interfere with the organization or stability of the SEC. Such inhibitors would be potential anticancer drugs for use against ACCs and other cancers, such as breast cancer, with overexpression of SEC components and in particular members of the AFF family.Two recently developed inhibitors (JQ1 and I-BET151) of bromodomain and extraterminal family members block cell proliferation of leukemia cell lines. They do so by indirectly interfering with the recruitment of SEC to chromatin from key cancer-related genes such as v-myc avian myelocytomatosis viral oncogene homolog (46 H295R stably transfected with the Tet repressor (H295R/TR) was kindly provided by Dr Lalli.47 H295R/TR/shRNA-βcatenin (shβ) clones carrying constructs encoding doxycyclin-inducible shRNAs targeting either CTNNB1 (βcatenin) are described in.12 The pSuperior.puro vector (Oligoengine), which can expresses a doxycyclin-inducible shRNA, was used to created the H295R/TR/shRNA-AFF3 clones. Silencing hairpin targeted AFF3 mRNA (targeted sequence: 5′-TAAGGACTCTCAGCTTGTA-3′) was cloned into pSuperior.puro vector following the manufacturer's instructions to give finally the pSuperior.puro/shRNA-AFF3 vector. H295R/TR cells were further transfected with the pSUPERIOR.puro/shRNA-AFF3 vector and clones were selected with puromycin . Three shRNA-AFF3 clones (shAFF3 clones) were selected in which AFF3 expression was downregulated at least 0.5-fold in a doxycyclin -dependent manner in comparison with control clone (Ctr) transfected with pSuperior.puro vector. All cell clones were investigated for their ability to express specific adrenocortical genes (StAR and CYP11B1). S45P CTNNB1 (β-catenin) gene activating mutation, previously identified in the parental H295R cell line, was confirmed by direct sequencing in all Ctr and shAFF3 clones (data not shown). The data presented are from a single Ctr clone and a single shRNA clone, but all results from in vitro experiments were confirmed by similar findings with three independent shRNA clones and two independent Ctr clones.ACC cells  and HeLa cells (ATCC CCL-2) were cultured, transfected and stimulated with staurosporin as previously described.AFF3 WRE (wild type or mutant) were tandemly inserted in the XhoI site of a pGL3Tk reporter plasmid, where luciferase expression is driven by the minimal Tk promoter. Double-stranded oligonucleotides with 5′ and 3′ XhoI overhangs were ligated in the open pGL3Tk vector. 2xWT-F, 5′-tcgaAGGAACAAAGGGGAGAGGAACAAAGGGGAG-3′ 2xWT-R, 5′-tcgaCTCCCCTTTGTTCCTCTCCCCTTTGTTCCT-3′ 2xMut-F, 5′-tcgaAGGGCCAAAGGGGAGAGGGCCAAAGGGGAG-3′ 2xMut-R, 5′-tcgaCTCCCCTTTGGCCCTCTCCCCTTTGGCCCT-3′. Inserted copy number was verified by DNA sequencing of the inserts. Rous sarcoma virus (RSV)-Renilla  was used as a control of transfection efficiencies. Cell tranfection was performed using effecten (Qiagen 301427). Firefly and Renilla luciferase activities were sequentially measured with the Dual Luciferase Reporter Assay System (Promega) and results are expressed as firefly luciferase activity normalized to Renilla luciferase activity of the same sample.The following small interfering RNA were used: siCTNNB1 (5′-AGCUGAUAUUGAUGGACAG-3′); siLEF1 (5′-CUACAGGAAUCUGCAUCAG-3′); siAFF3-1 (5′-AGAUGACCUUAAGCUAAGC-3′); siAFF3-2 (5′-UAAGGACUCUCAGCUUGUA-3′); si-FAM19A4 (5′-GGATGAGAGTCTGTGCTAA-3′); siISM1 (5′-AUAUCCAGGUCACCAUAGA-3′); siACPL2 (5′-GAAUGGAAUGAGUAGCAAG-3′); siAXIN2 (5′-UCAAGAAGCAGCAGAUUGA); siNKD1 (5′-GGAUGUGGCACAUAUAUAC-3′); siCtr (5′-AGCUGAUAUUGAUGGACAG-3′). The AFF3-Flag-tagged vector from origene  was used to overexpress AFF3. As a Wnt/β-catenin pathway reporter construct-driving expression of luciferase gene the Top plasmid was used, which contains two copies of the β-catenin/TCF-binding sites, whereas the Fop plasmid contains two mutated copies of the β-catenin/TCF-binding sites. Two copies of the putative CTNNB1: ; LEF1 ; AFF3 ; FAM19A4 ; ISM1 ; ACPL2 ; AXIN2 ; NKD1 ; PPIA (CYCLO) . The AFF3 variant mRNAs produced were analyzed by producing complementary DNA from total mRNA from H295R cells, then PCR using oligonucleotides  corresponding to sequences in exon 3 and exon 5, followed by agarose gel electrophoresisRNA extractions from cell lines were performed using Promega kit (Z6012). Reverse transcription of RNA were performed using High-capacity cDNA reverse transcription kit . The qPCR were performed using the light cycler 480 SYBR green . The PCR conditions for all target genes were: activation at 95 °C for 5 min followed by 45 cycles of 95 °C for 5 s, annealing at 60 °C for 5 s and 72 °C for 1 s/25 bases with the primer pairs (forward and reverse) described below: M Tris HCl (pH 7.4), 0,5% NP40, 20 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.8)) containing proteinase and phosphatase inhibitors (Roche) for 5 min at 4 °C. After centrifugation at 5000 g for 5 min, the supernatant was removed (cytoplasmic extract). The nuclear buffer (50 mM Tris HCl (pH 7.4), 1% NP40; 400 mM NaCl; 1 mM EDTA (pH 7.8)) containing proteinase and phosphatase inhibitors (Roche) was added to the pellet. Then samples were vortexed 15 s every 10 min for a total of 30 min at 4 °C. After centrifugation at 16000 g for 5 min, the balance buffer (50 mM Tris HCl (pH 7.4), 1% NP40, 1 mM EDTA) containing proteinase and phosphatase inhibitors (Roche) was added to the supernatant to make the final NaCl concentration 150 mM (nuclear extract). Flag immunoprecipitation: the nuclear extract was incubated with anti-Flag M2 affinity gel (Sigma A2220) overnight at 4 °C with gentle rotation. The affinity gel were spun down and washed three times with wash buffer (50 mM Tris HCl (pH 7.4), 1% NP40, 150 mM NaCl, EDTA 1 mM (pH 7.8)) containing proteinase and phosphatase inhibitors (Roche). The elution was made with the peptide 3X Flag (sigma F4799) at 4 °C for 30 min without agitation. CDK9 immunoprecipitation: the lysate was then incubated with antibodies and protein A coupled magnetic sepharose beads  overnight at 4 °C with gentle rotation. The beads were washed three times with wash buffer (50 mM Tris HCl (pH 7.4), 1% NP40, 150 mM NaCl, EDTA 1 mM (pH 7.8)) containing proteinase and phosphatase inhibitors (Roche) before boiling at 70 °C in 2% of sodium dodecyl sulfate-loading buffer for 10 min. Fluorescence immunostaining: H295R cells were washed with cold PBS once and were permeabilized using a solution of PBS with 0.5% Triton for 10 min. The cells were washed with PBS for three times before incubated with 10% normal goat serum blocking solution (Invitrogen 50-062Z) at room temperature for 1 h. The cells were then incubated with primary antibodies overnight at 4 °C. After three washes with PBS, secondary fluorescent antibodies were added to the cells for 1 h at room temperature. The nuclei were stained with 4′,6-diamidino-2-phenylindole and after mounting with Fluoromount G , images were taken with a fluorescence microscope (Zeiss Axiovert 200M). The following antibodies were used: Anti-cleaved Caspase3 (9661) and anti-LaminA/C (2032) , Anti-β-ACTIN , Anti-AFF3 , Anti-Flag (F1804) and anti-Sc35 (s4045) (Sigma-Aldrich), Anti-CDK9 (ab 38840) and anti-Cyclin T1 (ab 2098) , Anti-b-Tubulin (sc5274) and normal rabbit IgG (sc2027) , Alexa Fluor 488 Anti-mouse IgG (A11001), Alexa Fluor 488 Anti-rabbit IgG (A11008), Alexa Fluor 647 Anti-rabbit IgG (A21244), Alexa Fluor 647 Anti-mouse IgG (A21235) .H295R cells were washed with cold phosphate-buffered saline (PBS) once and lysed in a low-salt lysis buffer . Proliferation was measured with the MTT assay . For colony-formation assays, cells from each clone were seeded at low density in individual wells of a standard six-well plate and grown for 30 days in normal serum medium. Colonies were visualized by crystal-violet staining.r correlation test was used to evaluate the correlation between gene expression and AXIN2 expression in two, previously described, independent cohorts of ACC: Cohort 18 included 47 ACC and four NA (Gene Expression Omnibus data set GSE49280 and ArrayExpress data set E-TABM-311); and Cohort 2,10 33 ACC and 10 NA (Gene Expression Omnibus data set GSE33371). All samples were normalized in batches, independently for each chip type using the RMA algorithm (Bioconductor affy package), and probe set intensities were then averaged per gene symbol. Both final matrices were row-centered using the NA median. Differential expression was measured with moderated t-test (limma R package) and survival curves were obtained by the Kaplan–Meier method. Differences in survival were assessed with the log-rank test. Gene expression profiles for three H295R/TR/shβ and one control clones with and without doxycycline treatment were analyzed using Affymetrix Human Gene 1.0 ST arrays (ArrayExpress data set E-MTAB-3330) and with the moderated paired t-test (limma R package). All P-values were adjusted using the Benjamini–Hochberg correction method. All in vitro data reported with statistical analyses represent means from at least three experiments. The error bars indicate the s.d. Control conditions were set as one and data were analyzed by one way analysis of variance followed by Fisher's LSD posthoc test. Gene set enrichment analysis48 were performed with several data sets from breast cancers  data sets GSE6532, GSE4922 and GSE1456; and The Cancer Genome Atlas breast carcinoma data set) and with a data set with inhibition of CDK9 by Flavopiridol.40 Significance was set at P<0.05 (represented by * in figures); P<0.01 (**) and P<0.001 (***). Analyses were performed using R 3.0.3 with custom scripts, which can be provided on request.Pearson's Total RNA from H295R cells was extracted, and complementary DNA library was created using Epicentre ScriptSeq strand-specific after a RNA depletion optimized with the Ribo-Zero kit . A paired-end 10- bp sequencing was performed on a HiSeq 1500 device . Reads were mapped to the UCSC  reference transcripts from hg19 genome using RSEM (RNA-Seq by Expectation Maximization) and the Bowtie alignment software. Wiggle files in genomic coordinate were generated for visualization.


TAZ is a downstream agent of Hippo signal pathway. β-catenin is a cell adhesion molecule associated with the invasion and metastasis of carcinomas as well as a critical component of Wnt pathway. TAZ and β-catenin have long been thought to play a vital role in tumour development and progression. This study aimed to detect expression of TAZ and β-catenin in adenocarcinoma of the esophagogastric junction (AEG) and explore their clinicopathological significance.The expression of TAZ and β-catenin were detected by immunohistochemistry of 135 AEG samples, and analyzed with complete clinicopathological features. Overall survival rates were also calculated using the Kaplan-Meier method. Cox proportional hazard model was performed to assess the prognostic values. 37 normal mucosa and 41 dysplasia samples of esophagogastric junction (EGJ) were studied comparably.TAZ protein showed a strictly nuclear staining pattern in AEG and dysplasia with IHC. Expression of TAZ was higher in dysplasia and AEG compared with normal mucosa . The positive expression rate of nuclear β-catenin was significantly higher in carcinoma and dysplasia than that in normal mucosa . Abnormal expression rate of membranous β-catenin in AEG was significantly higher than that in normal mucosa tissues and dysplasia . In AEG, over expression of TAZ was directly correlated with abnormal nuclear β-catenin expression  and membranous β-catenin . Patients with abnormal TAZ or β-catenin expression of AEG exhibited a shorter overall survival (OS) and lower overall survival rate than those with normal TAZ or β-catenin expression (P < 0.05). In addition, patients with abnormal expression of both TAZ and β-catenin exhibited worst overall survival. In multivariate survival analysis, abnormal expression of TAZ, TAZ & β-catenin (nuclear and membranous) and tumour differentiation were found to be independent prognostic factors related to OS of AEG patients.Over expression of TAZ was associated with abnormal expression of β-catenin, which is correlated with poor prognosis of patients with AEG. Abnormal expression of TAZ and TAZ & β-catenin (nuclear and membranous) are independent prognostic factors, so targeting TAZ and β-catenin could prove to be a promising therapeutic strategy for the treatment of AEG.http://www.diagnosticpathology.diagnomx.eu/vs/2558852841276335The virtual slide(s) for this article can be found here:  Adenocarcinoma of the esophagogastric junction (AEG) is defined as carcinoma that crosses the esophagogastric junction (EGJ) line, irrespective of where the tumour epicenter is located, including both distal esophageal and proximal gastric carcinomas. In receThe transcriptional coactivator with PDZ-binding motif (TAZ), also called WWTR1 , was first reported as a 14-3-3 binding protein which isβ-catenin was originally identified as a membrane component of the cadherin-mediated cell-cell adhesion system and it is now widely recognized as a critical element of the Wnt signal pathway. In the The relationship between β-catenin and TAZ remains controversial. Some scholars believe that in the cytoplasm, the coactivator TAZ inhibits the CK1-mediated phosphorylation of Dvl2 by competing with Casein Kinase1 for combining with Dvl2, thereby inhibiting Wnt/β-catenin signalling and promoting the degradation of β-catenin. InhibitAlthough β-catenin or TAZ has been demonstrated to correlate with poor prognosis of a variety of malignancies, however, the correlation of TAZ and β-catenin expression in AEG and the relevance of their co-expression within clinical parameters still remain unclear. Moreover, the relationship between β-catenin and TAZ remains controversial.In this study, expression of TAZ and β-catenin was examined using immunohistochemistry on 135 AEG samples, compared with normal tissues and dysplasia samples. The correlation of TAZ and β-catenin expression and its relevance to clinicopathologic parameters were explored. Furthermore, the prognostic roles of TAZ and β-catenin in AEG were evaluated using Kaplan-Meier and Cox regression analysis. To the best of our knowledge, it is the first instance of reporting the correlation of TAZ and β-catenin expression and their clinical significance for patients with AEG.The samples were obtained upon receipt of informed consent from patients undergoing surgical resection. The study was approved by the ethics committee of Shandong University School of Medicine . Total samples comprised 135 cases of adenocarcinoma, 37 cases of normal mucosa tissues and 41 cases of dysplasia specimens which were located in EGJ. The cases were obtained from the archives of the department of Pathology at Qilu Hospital, Shandong University and collected from patients who underwent surgery between January 2006 and December 2007. The diagnosis was confirmed histologically in all cases, based mainly on examination of sections stained with H&E. Before surgery, no patients had received drug intervention and preoperative chemotherapy. Pathological characteristics were obtained from the medical records and the original pathology reports, including age, gender, tumour differentiation, tumour size, lymph node status and invasion of serosa. AEG cases were staged by TNM classification according to the standard for oesophagogastric junction tumours of American Joint Committee on Cancer (AJCC). Curative resection procedure was performed in all AEG patients and the resection boundaries were negative both in intraoperative and routine pathological diagnosis. The AEG patients were followed up by phone call. The total period of follow-up was 1–81 months (median was 35 months). The endpoint of this study was overall survival which is defined as the period lapsing from the date of initial biopsy until death or last follow. By October 2013, 89 patients were reached the end events.Immunohistochemical stains were performed on formalin-fixed, paraffin-embedded specimens with the PV-9000 2-step plus poly-HRP anti-mouse/rabbit IgG detection system  according to the manufacturer’s instructions. Briefly, 4 μm sections were cut and placed on glass slides, then dewaxed in xylene and rehydrated through graded alcohol. All the slides were boiled in EDTA antigen retrieval solution  for 3 min to retrieval the antigen. Thereafter, the sections were incubated with 3% hydrogen peroxidase (reagent A) at room temperature (RT) for 10 min to block endogenous peroxidase activity. After rinsing in phosphate buffered saline , incubated with normal goat serum  to block any nonspecific reactions for 10 min at RT. Shook off excess goat serum, and then incubated the slides with primary antibodies overnight at 4˚C, TAZ  and β-catenin  were diluted 1:100 and 1:200 in PBS. The sections were washed with PBS and incubated with polymer helper (reagent B) for 20 min. After rinsing in PBS, the sections were incubated with polyperoxidase-anti-mouse/rabbit IgG (reagent C) for 30 min at room temperature and DAB  was visualized. After rinsing in water, the sections were counterstained with hematoxylin, dehydrated, and coverslipped.Samples of AEG with high expression of TAZ and β-catenin served as the positive control. PBS was used instead of the primary antibodies as negative controls.TAZ and β-catenin immunostaining signals were evaluated independently by two pathologists in a blinded manner. Brown nuclear staining for TAZ was considered positive/abnormal. The staining intensity of positive tumour cells was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). The percentage of positively stained tumour cells was scored with 5 scales: 0 (<10 %); 1 (10% to 25%); 2 (26% to 50%); 3 (51% to 75%); 4 (>75%). The final score was the product of the intensity and the percentage. For statistical reasons, a final staining score ≥5 was considered to be positive. Nuclear and membranous β-catenin signals were evaluated independently. When more than 10% of cancer cells showed strong nuclear staining, the tumour was judged to exhibit positive/abnormal nuclear expression. In cases that demonstrated either no immunoreactivity at the membrane, or less than 10% of the tumour cells with positive membranous staining, the specimen was considered to abnormal membranous expression. The perT test. The correlation between TAZ and β-catenin was analyzed using the spearman’s rank test. The survival curves were estimated by the Kaplan–Meier method. Log-rank test was used to compare survival curves. The HR and the 95% CI were evaluated for each variable using the Cox univariate model. A multivariate Cox proportional hazard model was also developed using stepwise regression (forward selection) with predictive variables that were significant in the univariate analyses. P < 0.05 was considered to be statistically significant. All statistical analyses were carried out using SPSS software .Kruskal-Wallis text was used to assess the difference of TAZ or β-catenin expression in the three groups. The relationship between TAZ or β-catenin expression and clinicopathologic parameters were analyzed using Chi-square test and As shown in Figure The positive nuclear expression rates of β-catenin in normal mucosa, dysplasia and AEG were 18.9% (7/37), 41.5% (17/41) and 51.1% (69/135) and there was statistical difference among these groups . The expression of β-catenin in AEG and dysplasia were significantly higher than in normal mucosa  while no significant difference between AEG and dysplasia (P = 0.276).The abnormal membranous expression rates of β-catenin in the normal mucosa, dysplasia and AEG were 27% (10/37), 29.3% (12/41) and 57% (77/135), respectively. There was a significant statistical difference among the groups . Abnormal membranous expression of β-catenin in AEG was significantly higher than that in normal mucosa tissues and dysplasia , while no difference was found between normal mucosa tissues and dysplasia (P = 0.843).In AEG, interestingly, the statistical analysis revealed that TAZ expression positively correlated with nuclear β-catenin expression  95%, 0.136-0.462; P < 0.001)  were independent negative prognostic factors together with tumour differentiation. The result strongly suggests that TAZ and β-catenin may serve as disease prognosis indicators for AEG patient survival. Moreover, the regulation of TAZ on β-catenin expression provides a new molecular mechanism underlying TAZ promoted AEG progression and metastasis and may indeed suggest a feasible therapeutic strategy to inhibit AEG metastasis by targeting TAZ and β-catenin expressions.In conclusion, our research demonstrated that over expression of TAZ was associated with abnormal expression of β-catenin in AEG. We also provided convincing evidence that abnormal expression of TAZ or β-catenin was correlated with the poor prognosis of patients with AEG. Moreover, abnormal expression of TAZ and TAZ & β-catenin (nuclear and membranous) could be independent predictors of prognosis in AEG, so targeting TAZ and β-catenin could prove to be a promising therapeutic strategy for the treatment of AEG.AEG: Adenocarcinoma of the esophagogastric junction; EGJ: Esophagogastric junction; TAZ: Transcriptional coactivator with PDZ-binding motif; CI: Confidence interval; HR: Hazard ratio; OS: Overall survival.The authors declared that they have no competing interests.ZJ conceived of the study. SL participated in experiments, performed the statistical analysis and drafted the manuscript. CF and SW participated in case selection and followed up with AEG patients. All authors scored the immunostaining, read and approved the final manuscript.


The key role of the Wnt/β-catenin signaling in colorectal cancer (CRC) insurgence and progression is now recognized and several therapeutic strategies targeting this pathway are currently in developing. Wnt/β-catenin signaling not only dominates the early stages of sporadic colorectal cancer (SCC), but could also represent the connection between inflammatory bowel diseases (IBD) and increased risk of developing SCC. The knowledge on the sequential molecular events of Wnt-signaling cascade in IBD and during colorectal carcinogenesis, might provide new diagnostic/prognostic markers and could be helpful for optimizing the treatment protocols, thus improving the efficacy of Wnt-targeting therapies. We performed a comparative evaluation of the expression of some crucial molecules participating to Wnt signaling in an animal model of chemically-induced CRC and in human tissues obtained from patients suffering from IBD or at sequential stages of SCC. Specifically, we analyzed upstream events of Wnt signaling including β-catenin nuclear translocation and loss of E-cadherin and APC functions, and downstream events including c-Myc and Cyclin-D1 expression. We demonstrated that these crucial components of the Wnt/β-catenin pathway, when evaluated by immunohistochemistry using a multiparametric approach that includes the analyses of both expression and localization, could be potent markers for diagnosis, prevention and therapy in IBD and SCC, also possessing a predictive value for responsiveness to Wnt-targeting therapies. Furthermore, we showed that the animal model of chemically-induced CRC mimics the molecular events of Wnt signaling during IBD and SCC development in humans and may therefore be suitable for testing chemopreventive or therapeutic drugs targeting this pathway. Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the developed countries , where iIn the last years, great attention has been given to the so called biological therapy, including innovative therapies employing cells, monoclonal antibodies or specific inhibitors of important steps of cell proliferation and transformation. These therapies are based on the knowledge of molecular pathways involved during cancer development and on the discovery of new specific molecular targets. Drugs directly acting on components of the signaling pathways implicated in tumorigenesis have exhibited clinical benefit in patients with various tumor types, including CRC -14.Therefore, deepening of knowledge on the molecular pathways actively involved in CRC insurgence and progression could potentially provide new targets for drug delivery and therapy, allowing to overcome the poor response to the current therapeutic strategies. Crucial molecules of these signaling should be also validate as new CRC-related biomarkers for the improvement of the current diagnostic/prognostic tools. Moreover, it is recognized that CRC represents a complication of inflammatory bowel diseases (IBD) and patients with long-standing IBD have an increased risk of developing CRC. Many of the molecular alterations responsible for sporadic colorectal cancer (SCC) also seem to play a role in colitis-associated colon carcinogenesis . Thus, kIn this context, the possibility to induce colon tumors in animals provides the opportunity to study various aspects of the carcinogenetic process, including detailed information on the subsequent events leading to the formation of neoplastic lesions. In particular, animal models of CRC could be useful to analyze the expression of novel markers of tumor development during colorectal cancer insurgence, progression and metastases and to identify possible targets for new chemopreventive and/or therapeutic drugs.In the last decade, numerous studies demonstrated the key role of deregulation or constitutive activation of the Wnt pathway in the initiation and progression of different forms of human cancer, including CRC -18. HencIn this study we analyzed, by immunohistochemistry, the expression of some crucial molecules involved in the signal transmission initiated by Wnt ligands, in colorectal inflammatory disease and during the CRC carcinogenetic process. Specifically, we analyzed upstream events of Wnt signaling including β-catenin nuclear translocation and loss of E-cadherin and APC functions, and downstream events including c-Myc and Cyclin-D1 nuclear expression. We performed a comparative evaluation of these molecules in an animal model of chemically-induced CRC and in human tissues obtained from patients suffering from IBD or at sequential stages of SCC, from low grade dysplasia to advanced carcinoma. The animal model used in this study consisted in syngenic immunocompetent BDIX rats, set up in our laboratory, in which colorectal adenocarcinoma was chemically induced by 1,2-dimethylhydrazine (DMH) administration. The DMH rodent models are widely used to study chemically-induced colon cancer , 26, andth week after the first DMH-administration, IBD-like morphological modifications were recovered, with crypt architectural distortion, inflammatory infiltrations in the lamina propria and basal plasmacytosis  and submucosal invasion through the muscularis mucosa  for each carcinogenetic stage vs NM and vs the previous stage are reported in Supplementary Colorectal tissues explanted from rats and classified in the different histopathological groups  were compared with human tissues, obtained from patients suffering from IBD and at the sequential stages of sporadic colorectal cancer, for the expression of the Wnt pathway components reported in the scheme of Figure vs nuclear β-catenin, that was observed only in 1 out of 5 samples, with 10-50% of positive nuclei. β-catenin localization at the cell membrane was also observed in the 100% of the rat tissues histologically classified as K, but, differently from humans, in rats all 5 samples also showed nuclear and cytoplasmic staining.In all samples of normal mucosa from humans and rats, β-catenin staining localized at the cell membrane, while cytoplasmic and nuclear staining was generally absent Figure . In humavs NM, quantified by assigning a score for each pattern of staining intensity and subcellular localization , with nuclei almost completely negative except for few c-Myc+ Figure , that lo+ Figure . In humae Figure , while Ce Figure .vs NM , and in human specimens from patients suffering from IBD (Chron's disease or ulcerative rectocolitis).Owing the role recently ascribed to Wnt-pathway activation in IBD-associated carcinogenesis , we finavs NM, with positive nuclei observed in 10-50% of colonic epithelial cells   Figure .vs NM from humans and rats , and in all samples analyzed this onco-protein localized in the nucleus of 10-50% of colonic epithelial cells (4 out of 5 samples) or in more than 50% of cells (1 sample)  Figure .We finally combined the quantitative results obtained on human samples from IBD to K stages  to LD, HD, IS and K . Moreover, the comparative evaluation of ß-catenin, E-cadherin, APC, c-Myc and Cyclin-D1 in rat and in human, demonstrated that our preclinical model, although minor differences, mimics the molecular events of Wnt signaling during IBD and SCC carcinogenesis in humans. Indeed, we recorded, already in IBD and in early stages of neoplastic transformation (LD and HD), changes in the expression and tissue distribution of these molecules, indicative of Wnt/ß-catenin pathway activation, while Cyclin-D1 was modified at later stages, in IS and, even more, in K. Indeed, the Wnt/ß-catenin pathway have an active role from IBD to IS, with upstream and downstream events of Wnt signaling wholly activated at the IS stage, while in advanced carcinoma the role of this pathway seems to overshadow, as indicated by the absence on nuclear ß-catenin.The first result that we obtained in this study was the validation of our pre-clinical model of CRC. As in other DMH-induced rodent model, in our rat model of colorectal cancer, carcinogenesis develops through a multistep process similar to human colon carcinogenesis Figure . In thisThe most important and innovative result concerns the design of a multiparametric panel obtained by combining both expression and subcellular localization of all molecules analyzed Figure . By assiIn conclusion, we demonstrated that crucial components of the Wnt/β-catenin pathway, when evaluated by immunohistochemistry using a multiparametric approach that includes the analysis of both expression and localization, could be potent markers for diagnosis, prevention and therapy in IBD and sporadic colorectal cancer, also possessing a predictive value for responsiveness to Wnt-targeting therapies. Even if the biomarkers included in this study should be validated with a larger number of samples to increase the statistical significance, our results provide the basis for the development of a multiparametric platform that might be used to design “specific maps” for CRC early diagnosis and staging, and that would represent an useful tool for determining the responsiveness to new drugs targeting the Wnt signaling. Even if clinically useful agents that specifically inhibit Wnt signaling cascade are not currently available, several therapeutic strategies in this sense are in currently developing and need to be validate , 34-39. n=5) were used as control group to obtain normal colonic mucosa. DMH-treated rats  were sacrificed, by inhalation of an overdose of CO2, once every two week, starting from the 6th to the 30th week after the first DMH administration. At autopsy, the entire colon rectum was excised, flushed with saline, pre-fixed in 10% buffered formalin for 2h and cut transversally in ring-shaped pieces of about 1mm thick. After additional 22h of formalin fixation, samples were embedded in paraffin in a routine manner for histological and immunohistochemical analyses. Sections from each paraffin block of rat or human tissues were stained with H&E for the histological examination.Inbred male BDIX rats , 8 weeks old and weighing 220–250g, were housed for 7 days, in a pathogen-free animal facility with free access to water and food in accordance with European Community guidelines. Experiments were approved by the local committee on animal experimentation, and were performed under strict governmental and international guidelines. Following a 1-week acclimation period, and after rat anesthetization by inhalation of 1-bromo-2-chloro-1,1,1-trifluoroethane , carcinogenesis was induced by DMH administered subcutaneously at the dose of 30mg/kg, once a week for 5 weeks. Untreated animals (n=30), taken for diagnostic purposes or after surgical excision of localized disease, were collected from pathology archives and analyzed anonymously in adherence to ethical standards. In this study we used the following samples: 5 specimens from patients suffering from IBD (Chron's disease or ulcerative rectocolitis), 5 from low grade dysplasia (LD), 5 from high grade dysplasia (HD), 5 from in situ carcinoma (IS) and 5 from advanced carcinoma (K). Normal colon biopsy specimens  were obtained from patients in whom colonoscopy was performed to rule out colorectal disease. The diagnosis was based on histopathological examination of routinely processed tissue together with clinical and laboratory data. Serial sections from each sample were subjected to hematoxylin-eosin (H&E) staining or to immunohistochemical analyses as described below.Paraffin-embedded human colon specimens  or included within the different histopathological groups . For immunohistochemical analyses, serial sections from rat or human tissues were collected on SuperFrost® Plus coated slides  and immuno-stained for ß-catenin, E-cadherin, APC, c-Myc and Cyclin-D1, using the specific antibodies and the working dilution reported in Supplementary the percentage of samples in which positive cells were less than 10% (negative or weakly positive), from 10 to 50%, and more than 50% of the cells constituting the epithelial mucosa;vs normal mucosa (NM) has been also reported as fold vs NM, calculated by the formula:the staining intensity  and the percentage of positive cells , as compared to the background control (Ctr II step), following the criteria reported in Table For the multiparametric analysis of all molecules analyzed, we defined the following cut-off for negative, positive and low-positive results, based on the arbitrary score assigned for each staining pattern of expression and localization Table :negative: biomarkers with mean score values ≤ 6, i.e. the sum of score assigned to −/± intensity (score=1) plus score assigned to % of positive cells ˂10% (score=5)positive: biomarkers with mean score values ≥10low-positive: biomarkers with mean score values ˃6 and ˂10t test and a P value threshold of < 0.05. Data of the mean scores obtained for each biomarker were presented as mean ± SD. For statistical analysis, groups were compared using the two-tailed Student's


CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease.Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge “maps” of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase  A wealth of knowledge relevant to the biological mechanisms of human disease, including information on protein-protein interactions (PPIs), protein post-translational modifications (PTMs), gene/protein expression and disease-associated mutations is contained in the scientific literature and bioinformatics databases. While it is challenging to collect information related to a gene/protein or disease of interest that is scattered across the scientific literature and housed in specialized databases with incompatible formats, development of systematic workflows to integrate and analyze information from disparate sources has the potential to uncover missing links and lead to new insights into disease etiology and treatment.Combination of text mining tools to extract information from the scientific literature, curated databases, and ontologies, which enable structured representation of entities, relations, and concepts is a powerful strategy for knowledge integration. In previous work , we deveCTNNB1) is a multi-functional protein that serves as both a cell adhesion molecule and transcriptional co-activator [GSK3B, creates a recognition site for the ubiquitin ligase BTRC, which ubiquitinates beta-catenin, targeting it for degradation by the proteasome. Dysregulation of beta-catenin activity is strongly correlated with cancer. Mutations that lead to beta-catenin disassociation from adherens junction and escape from ubiquitin-mediated degradation result in its translocation to the nucleus where it hyper-activates the transcription of its target genes, several of which have oncogenic activity [Beta-catenin , which provides a unified presentation of PTM information text-mined from the scientific literature and from multiple high-quality curated databases, including PhosphoSitePlus [Extending our previous work, we developed a bioinformaticsframework to capture and integrate important molecular relations and attributes for proteins of interest for construction of knowledge maps . The oveSitePlus —and PhosSitePlus . To deveA network of beta-catenin molecular relations is shown in LEF1, has been shown to participate in a positive transcriptional feedback loop with beta-catenin. In colon cancer cells, beta-catenin/LEF1 complexes drive transcription of the full-length LEF1 isoform that can bind beta-catenin at the expense of a dominant negative isoform. The expressed LEF1 then associates with beta-catenin to drive further expression of full-length LEF1 [Proteins within the network that participate in more than one molecular relation are of special interest because they can provide insight into the regulation of beta-catenin and the coordination of its multiple functions. To demonstrate this idea, we used the beta-catenin network to identify proteins that might be involved in transcriptional feedback loops with beta-catenin. Feedback mechanisms, in which the product of an expressed gene stimulates (positive feedback) or suppresses (negative feedback) the transcriptional program that controls it, play a critical role in cellular transcriptional regulation. For example, the transcription factor gth LEF1 .As outlined in LEF1, a member of the TCF/LEF family transcription factors; the androgen receptor AR; the CCAAT/enhancer binding protein CEBPA; the estrogen receptor ESR1; and the nuclear factor-kappa-B p105 subunit (NFKB1). With the exception of LEF1, these transcription factors are not completely dependent on beta-catenin for activity; they can work with other co-regulators or initiate their own transcription. Thus, we consulted the literature to determine what is known about the contribution of beta-catenin to the transcription of the target-interactors we identified. Fifteen of the target-interactors plus beta-catenin itself have either been shown to be regulated by beta-catenin in small-scale studies (the Wnt Homepage (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes); Herbst et al., Table 1 [LEF1-regulated target-interactors, and also multiple targets of ESR1, NFKB1, and CEBPA ) were up-regulated by beta-catenin and two (FOS and CDH1) were down-regulated; for the remaining genes, the directionality of the beta-catenin effect was not reported.Five different transcription factors regulate the expression of the target-interactors:  Table 1  or were  Table 1 . This grNot all proteins that interact with beta-catenin necessarily affect its transcriptional activity. Therefore, the next step toward identifying potential transcriptional feedback mediators was to determine what effect, if any, the target-interactors had on the transcription regulation function of beta-catenin . We searTCF3, TCF7L1, and LEF1), AR, and MITF, which controls transcription of melanocyte-specific genes [The target-interactors that potentially increase beta-catenin transcriptional regulatory activity include beta-catenin itself and several transcription factors co-regulated by beta-catenin: three TCF/LEF family members . Moreover, Tyr-142 phosphorylation decreases beta-catenin association with the adherens junction, by inhibiting binding to alpha-catenin (PR:000036860), respectively. Reduction of association with the adherens junction increases the pool of beta-catenin available for transcriptional regulation in the nucleus. Similarly, two other target-interactors—MET and MUC—dissociate beta-catenin from the adherens junction and promote its translocation to the nucleus [Two beta-catenin kinases— nucleus ,21.GSK3B phosphorylates beta-catenin on N-terminal sites (PR:000035772) that promote its association with the ubiquitin ligase BTRC and subsequent degradation); ii) via interaction with “inhibitors” in the nucleus  protein in the nucleus [CDH1) associates with beta-catenin at the adherens junction [CDH1 transcription, CDH1 sequestration of beta-catenin away from the nucleus may actually result in an increase in CDH1 expression.The target-interactors that decrease beta-catenin transcriptional regulatory activity act via several mechanisms: i) by promoting beta-catenin degradation ; and iijunction ). It is CSNK2A1 and CSNK2A2, GSK3B.Interestingly, casein kinase II  knock-down screens have been performed to understand the impact of kinase signaling pathways on beta-catenin activity and sub-cellular distribution. One such study identified a group of kinases that seems to positively regulate beta-catenin co-activator activity under normal conditions . One of survival . Recentl-catenin . CDK5, l-catenin . Activat-catenin . Finally-catenin . Thus, wCDK5 phosphorylates beta-catenin on Ser-191 and Ser-246 (PR:000037229); however, the effect of this phosphorylation on beta-catenin transcriptional activity has not been reported. While it remains possible that CDK5 directly regulates beta-catenin transcriptional activity, we looked for evidence that CDK5 might act indirectly through phosphorylation of another protein in the beta-catenin biological network. The workflow for this analysis is presented in EGFR and ERBB2. Moreover, rat ERBB2 is reported to be a CDK5 substrate in PhosphoSitePlus [ERBB2 can also be phosphorylated by CDK5. These findings raise the possibility that CDK5 can indirectly regulate the phosphorylation of beta-catenin through its influence on other beta-catenin kinases.SitePlus . Human aCDK5 and the kinases it regulates leads to the production of five phosphorylated proteoforms: a Tyr-654 phosphorylated form ; a Tyr-333 phosphorylated form ; a Tyr-86/Tyr-654 doubly phosphorylated form ; a Ser-191/Ser-246 doubly phosphorylated form ; and a Ser-663/Ser-675 double phosphorylated form . With the exception of the Ser-191/Ser-246 phosphorylated form, PRO annotation indicates that all of these forms are transcriptionally active. We used RLIMS-P to identify sentences in the literature describing phosphorylation of ERBB2, SRC, and PAK1 by CDK5 and manually reviewed the article sections containing the sentences to determine the impact of CDK5 phosphorylation on substrate activity. Two of the kinases—PAK1 and SRC—are inhibited by CDK5 and one—ERBB2—is activated [CDK5 may tend to decrease beta-catenin co-activator function through its effects on PAK1 and SRC and increase co-activator function through its effects on ERBB2. The net effect of CDK5 will depend on the levels of these three kinases, their relative affinities for beta-catenin and the cellular context. The fact that CDK5 promoted beta-catenin transcriptional activity in the siRNA screen suggests that its role in ERBB2 activation may predominate. Interestingly, CDK5 has been shown to weaken beta-catenin association with the adherens junction by promoting the phosphorylation of Tyr-654 [ERBB2 phosphorylation site on beta-catenin, this result is consistent with our hypothesis that CDK5 activates ERBB2, which in turn phosphorylates beta-catenin on Tyr-654, leading to a shift of beta-catenin away from the adherens junction and into the nucleus where it can serve as a transcriptional co-activator. Moreover, in cancer cells, CDK5, ERBB2/ERBB3, and beta-catenin have been linked through another member of the beta-catenin network—the androgen receptor (AR). In ERBB2-overexpressing breast cancer cells, beta-catenin co-activates AR to drive transcription of several tumor-promoting targets, including ERBB3 {{}}[AR activation by CDK5 phosphorylation has been identified as a cancer-driving mechanism in prostate tumors [ERBB2/ERBB3 and AR could drive a feedback loop, in which ERBB2/ERBB3 promotes beta-catenin transcriptional activity that then contributes to higher expression of ERBB3. By combining the kinase and substrate information in multiple phosphorylation databases with phosphorylation-focused mining of the scientific literature, we identified a pathway linking beta-catenin to an upstream kinase, CDK5, shown to influence beta-catenin co-activator activity in a kinome-wide siRNA screen.We next conducted an in-depth study of the relationships between CDK5, ERBB2, ERBB3, SRC, PAK1, and beta-catenin based on text mining results and PRO functional annotation. As shown in ctivated –30. Thus Tyr-654 . Since TRBB3 {{}}. As mente tumors . Taken tGSK3B phosphorylation sites  and two highly conserved residues in the BTRC ubiquitin ligase recognition motif (Asp-32 and Gly-34). Several proteoforms phosphorylated on different combinations of the four highly mutated phosphorylation sites have been described in the literature . The top six most frequently mutated sites across all cancer types, accounting for 6–25% of all cancer-associated mutations in beta-catenin . Since the overall frequency of mutations of these sites is similar we assume similar frequency of mutation in the sampled tissues. However, we found two clusters with highly distinct mutation profiles emerged from this analysis . ClusterBTRC binding sites, may be driven by loss of BTRC binding and beta-catenin stabilization as the canonical model proposes. However, these tissues have a low mutation frequency at the priming phosphorylation sites, Thr-41 and Ser-45. Assuming that Thr-41 and Ser-45 mutations occur in these tissues at the same rate as in the overall set, the relative absence of these mutations in cancer samples suggests that these mutations are not strong cancer drivers in these tissues. Thus, phosphorylation of Ser-33 and Ser-37 may be independent of upstream priming phosphorylation events. A beta-catenin proteoform phosphorylated on Ser-33 and Ser-37 capable of binding BTRC has been reported; this form is phosphorylated by HIPK2 kinase, which does not require upstream priming . Taken together, these results suggest that the high frequency of Ile-35 and His-36 mutations in cancers is likely to reflect impairment of BTRC binding to beta-catenin.Eight additional residues in the beta-catenin exon 3 region each account for at least 0.5% of the total cancer-associated mutations. Two of these residues—Ile-35 and His-36—lie at less conserved positions of both the In this paper, we presented a beta-catenin knowledge map generated by capturing information about PTM, PPI, transcriptional co-activation and transcriptional targets, as well as beta-catenin disease-associated mutations, sequence features, and functional information. Publicly available text-mining and data-mining tools enabled us to integrate and analyze this wide variety of knowledge. Our approach helped us to gain insight into the roles of beta-catenin in healthy and cancerous cells.Such knowledge integration can support scientific research and lead to novel discoveries, as illustrated by our beta-catenin study. First, it is a mechanism for organizing information about a topic that is otherwise scattered, providing a comprehensive, integrated picture of what is known about a protein. For example, it is possible to easily identify proteins that are connected to the protein of interest by more than one type of relation. In our study, we analyzed proteins that were both (i) beta-catenin interacting proteins and (ii) potential beta-catenin transcriptional targets . These are proteins whose expression may be regulated by beta-catenin that, in turn, modulate beta-catenin activity in some way. By looking at the effects that these proteins have on beta-catenin transcription function, we found several candidates that may participate in positive or negative transcriptional feedback loops. Because transcription factor-target relations and PPIs are most often reported in separate articles or stored in separate databases, proteins that share both relationships are not readily apparent without knowledge integration.CDK5, the ERBB2/ERBB3 receptor tyrosine kinase, and beta-catenin that could explain the inhibitory effect of CDK5 siRNA knock-down on beta-catenin co-activator activity. The individual pieces were already known: CDK5 was known to positively regulate ERBB2/ERBB3 by phosphorylation; ERBB2/ERBB3 were known to phosphorylate beta-catenin on Tyr-654; and Tyr-654 phosphorylation was known to stimulate beta-catenin co-activator function at the expense of its cell adhesion function. However, viewing these pieces side-by-side made it possible to see how they could fit together into a single pathway.Additionally, we can generate hypotheses to explain experimental findings, pointing the way to future experiments. For example, the development of high-throughput siRNA-based knock-down screening methodologies has produced data on gene product-phenotype connections in mammalian cells on a scale that was previously possible only in simpler model organisms. However, as in classic mutational screening approaches, extensive follow-up studies are often necessary to understand the molecular mechanism by which the knock-down of a gene product leads to the observed cellular phenotype. A biological network can be used to explore knowledge gaps and guide these follow-up studies by identifying pathways that connect the perturbed gene product with molecules that could be mediating the phenotype. Using our beta-catenin data, we proposed a chain of events linking Finally, examining beta-catenin mutations across cancer types and at particular residue positions (e.g. Ile-35 and His-36) revealed interesting patterns. By overlaying this information with beta-catenin sequence features and PTM proteoform-specific functional information, we were able to propose explanations for how the different mutation patterns could contribute to dysfunction of beta-catenin and disease. Ontologies such as PRO, which supports the definition of PTM proteoforms and GO, which provides a structured format for functional annotation, were particularly helpful for the interpretation of mutations in the N-terminal phosphorylation sites of beta-catenin because we could easily see which proteoforms, and which associated beta-catenin functions were affected in each cluster of cancers.Knowledge integration poses several challenges. One concern is quality control of the integrated data. The quality of the constructed networks and the scientific conclusions drawn from then depend heavily on the quality of the molecular interactions that comprise them. Many of the resources used to build our knowledge network, such as STRING and the Transcriptional Regulatory Element Database (TRED), employ a scoring system for assigning confidence to the relations they contain. For our beta-catenin network, we set moderate cutoff scores  to strike a balance that limited inclusion of inaccurate relationships while still creating a comprehensive view of the cellular roles of beta-catenin. In general, confidence cut-offs can be set more or less stringently depending on user needs. We also address the issue of data quality by using manually curated databases. Currently, we manually verify all of our text-mining results; however, we are working toward further automation by developing systems to assign confidence to relations extracted by text-mining. These systems will take into account the article section where the relation is found  as well as the language used by the authors in stating the relations .Another challenge is that most resources express molecular interactions in terms of gene-level entities using the gene symbol or UniProtKB accession number to identify the participants, thereby obscuring any proteoform-specific effects. PRO, which provides a framework for representing the multiple proteoforms that arise from a single gene, can play a key role in addressing this issue. In this study, we have associated PTM proteoforms of beta-catenin defined in PRO with their modifying enzymes and with proteoform-specific functional information. Going forward, usage of text-mining tools will be valuable for retrieving in depth information about proteoform-specific molecular relations. For example, we have developed the Extracting Functional Impact of Phosphorylation (eFIP) text-mining tool to detect effects of phosphorylation on PPI; extension to other impacts, such as changes in enzymatic activity are planned .http://proteininformationresource.org/iPTMnet/). Using information text-mined from the literature as well as information in curated PTM resources, iPTMnet links enzyme-substrate relationships, ontologies, and functional impacts, such as PPIs, and provides a visualization of these PTM relationships. Finally, the ability to conduct relation-centric (as opposed to entity-centric) searches allows users to explore the relations that connect their genes/proteins of interest. By linking together multiple relation-centric searches, genes/proteins connected by complex relation types can be identified. Most other integrated resources such as UniProtKB [The time and effort required to extract information from the disparate resources used in this study highlighted the desirability of creating an integrated query interface that enables browsing, searching, and visualization of a wide range of gene, protein, disease, and drug information from a single web portal with knowledge derived from multiple sources. Toward that end, we are continuing to develop our iPTMnet database and web portal  and in files downloadable as part of PRO release 45 (April 2015).Information about phosphorylation of human beta-catenin was retrieved from the literature by querying the text mining tool, RLIMS-P  with thehttp://dbptm.mbc.nctu.edu.tw/) [http://proteininformationresource.org/iPTMnet/), which integrates information from PhosphoSitePlus (http://www.phosphosite.org/) [http://phospho.elm.eu.org/) [http://www.hprd.org/) [Additional information about beta-catenin PTM sites, PTM enzymes, and PTM enzyme binding motifs was obtained from dbPTM (edu.tw/)  and our te.org/) , Phosphoeu.org/) , and Humrd.org/) , as wellhttp://cbrc.kaust.edu.sa/tcof/) [AR, ESR1, CEBPA, NFKB1, and two TCF/LEF family members (LEF1 and TCF7L2), due to the evidence that they are co-activated by beta-catenin. Target genes of for five of these transcription factors  were obtained from TRED; http://rulai.cshl.edu/cgi-bin/TRED/; parameters: Binding Quality—1:Known, Promoter Quality: 1: Known-curated, known, 2: Refseq-predicted and 3: Refseq); no target genes were available for TCFL2 in TRED. We downloaded human beta-catenin interacting proteins from the STRING database v.9.05  [http://cancer.sanger.ac.uk; [To identify the transcriptional factors co-activated by beta-catenin, we used Dragon Database of Transcription Co-factors and Transcription Factor Interacting Proteins  . Of ninee > 0.6) . Informar.ac.uk; .Network views were created using Cytoscape v3.1.1 .http://david.abcc.ncifcrf.gov/) [http://www.r-project.org/) treemap function. In the treemap, supercategories (colored blocks) correspond to functional clusters; the sizes of the individual term boxes are determined by the p-values for the enrichment.Functional annotation clustering of GO Biological Process terms was performed using the DAVID web interface (rf.gov/)  with defhttp://www.r-project.org/) with default parameters. For the analysis of Ile-35 and His-36 substitutions Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Some degree of external root resorption is a frequent, unpredictable, and unavoidable consequence of orthodontic tooth movement mediated by odontoclasts/cementoclasts originating from circulating precursor cells in the periodontal ligament. Its pathogenesis involves mechanical forces initiating complex interactions between signalling pathways activated by various biological agents. Resorption of cementum is regulated by mechanisms similar to those controlling osteoclastogenesis and bone resorption. Following root resorption there is repair by cellular cementum, but factors mediating the transition from resorption to repair are not clear. In this paper we review some of the biological events associated with orthodontically induced external root resorption.  Some apical external root resorption is an invariable side effect of orthodontic treatment. It affects most frequently the maxillary incisors and is associated with several biological and mechanical risk factors  1–11].–11.1–11]It is caused by orthodontic load-induced sterile inflammation that brings about resorption of the superficial root cementum, or it can become more severe with eventual resorption of the underlying dentin . Apical Intrinsic factors that may play roles in the pathogenesis of orthodontic load-induced apical external root resorption include polymorphism of genes encoding cytokines and growth factors, alveolar bone density and turnover, hormonal deficiencies, and other local anatomical factors , 12–16.Reportedly, in about 80% of subjects, teeth undergoing orthodontic treatment may develop some degree of apical external root resorption . It is aMany studies have been carried out on laboratory animals, and the results of such studies, although providing important information on the pathogenic mechanisms of apical external root resorption, cannot be reliably extrapolated to the outcome of treatment of patients , nor canNevertheless, it is estimated that up to 90% of orthodontically treated teeth have some degree of apical external root resorption, and up to 15% of these cases show severe apical resorption of more than 4 mm  5, 7, 1, 17, 17.Since the area of periodontal attachment of a tapering root per millimeter of root length decreases very substantially from the cervical towards the apical region, it is estimated that the loss of periodontal attachment from 3 mm of apical external root resorption is equivalent to about 1 mm of cervical bone loss . Indeed,Cementum is the thin layer of mineralized tissue at the root surface providing anchorage for the principal fibers of the periodontal ligament , 27. CemDespite the fact that the differentiation of both cementoblasts and osteoblasts is driven by bone morphogenetic proteins (BMPs), it is unclear whether cementoblasts and osteoblasts are derived from a common mesenchymal multipotential precursor cell , 27 or wWhile in many aspects cementum is similar to bone, cementum differs from bone in having only a limited capacity for remodelling, in lacking innervation, vascularization, or lamellar structure, and in lacking any role in calcium homeostasis or haemopoiesis , 30–32; Cementum resorption and subsequent repair by cellular cementum (cementum turnover), which according to some researchers can be regarded as a physiological remodelling process, occurs throughout life in response to metabolic changes in the periodontal ligament mediated by forces of mastication, by continual tooth eruption and drifting, and possibly by parafunctional gnathic activities , 32–34. β-catenin and in the receptor activator of nuclear factor κβ (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) signalling pathways. Under these circumstances, the extent of the resorption is influenced by the degree of dysregulation of these pathways [Resorbed acellular cementum is always repaired with cellular cementum but the molecular pathways and cellular mechanisms which mediate this process are unknown , 33. Extpathways .The site of orthodontically mediated resorption on a root surface is determined primarily by whether the tooth is moved bodily or is tipped and occurs predominantly on those parts of the root surfaces exposed to high compressive stresses ; but it Bodily tooth movement generates compressive stresses along root surfaces with resorption of cementum at zones of compression of the periodontal ligament, but such resorption is less frequent and less severe than the apical root resorption associated with tipping tooth movements  because Furthermore, the thinner, more elastic alveolar bone around the cervical one-third of the root has the capacity to absorb much more of the orthodontically induced mechanical stress compared to the thick, less elastic alveolar bone that envelops the apical one-third of the root , 38, 39.Orthodontically induced root resorption can be evaluated by conventional radiography, subtraction radiography, cone-beam computed tomography, scanning electron microscopy, and histopathology , 20. ConIn general, in both bodily and tipping tooth movements, external root resorption increases with the magnitude of the applied orthodontic force , 19, 41 In the context of orthodontic tooth movement, the necrosis and hyalinization in the periodontal ligament and alveolar bone occur in response to localized orthodontic load-induced compression of the vasculature and subsequent ischaemia in the periodontal ligament and adjacent alveolar bone , 37, 43.During load-induced resorption of cementum, it appears that, as in the case of osteoclast differentiation and function , a numbeCementoclasts/odontoclasts have a phenotype similar to that of osteoclasts , being mAfter the cementoclasts/odontoclasts have become detached from the resorption lacunae, early cementum repair starts with fibroblast-like cells from the periodontal ligament invading the resorption lacunae . These fβ (TGF-β) also play important roles in cementoblast precursor differentiation and cementoblast proliferation, further promoting cementogenesis [During this process of cementum repair, extracellular matrix proteins from resorbed cementum including fibronectin, osteopontin, and osteocalcin contribute to the recruitment of cementoblast precursors to the root surface and to their subsequent adhesion, proliferation, and differentiation. Local cytokines and growth factors including insulin-like growth factor- (IGF-) 1, fibroblast growth factor (FGF), epidermal growth factor (EGF), bone morphogenetic proteins (BMPs), and transforming growth factor-ogenesis .β-catenin signalling pathway can promote epithelial-mesenchymal transition with dedifferentiation of the epithelial cell rests of Malassez which subsequently differentiate into cementoblast-like mesenchymal cells that can contribute to the formation of reparative cementum [As mentioned previously, epithelial cell rests of Malassez in the periodontal ligament may also play some role in the formation of reparative cementum , 52. Uprcementum , 52. It β-catenin signalling pathway, in addition to its role in epithelial-mesenchymal transition in the context of cementogenesis, is essential for osteoblast differentiation during embryogenesis and postnatally for regulating osteoblast proliferation. Canonical Wnt/β-catenin signalling is characterized by accumulation of β-catenin in the cytoplasm and subsequently its translocation into the nucleus of both osteoblasts and cementoblasts where it interacts with members of the Lef/Tcf family of transcription factors [The Wnt/ factors .β-catenin signalling is regulated by a balance between agonists of frizzled receptors and LRP 5/6 coreceptors on one hand and several antagonists including Dkks and sclerostin on the other hand [β-catenin in mature osteoblasts may induce the production of osteoprotegerin resulting in inhibition of differentiation and of functional activity of osteoclasts. Thus, the canonical Wnt/β-catenin signalling pathway via different vectors plays an essential role in bone turnover and subsequently in the regulation of bone mass [Wnt/her hand . In addione mass .Runx 2 is the master transcription factor that upregulates the genes ALP, collagen type 1, osteocalcin, and osteopontin that mediate osteogenic differentiation . Runx 2 β-catenin signalling pathway in cementoblasts inhibits the expression of Runx 2 and osterix, resulting in downregulation of cementoblast differentiation and proliferation [β-catenin signalling pathway is positively associated with differentiation of cementoblasts and subsequent increased formation of cementum [β-catenin signalling is associated with increased cementum formation [β-catenin signalling is associated with increased resorption of cementum [β-catenin signalling pathway plays an important role in the differentiation of cementoblasts and in the formation of cementum [In relation to cementogenesis, the activated Wnt/feration . Converscementum . Howeverormation ; but dowcementum . In any cementum , 57.β-catenin signalling in cementoblasts and osteoblasts, probably via upregulation of DKK1 expression, osterix inhibits osteoblast and cementoblast proliferation [Osterix is a transcription factor essential for the differentiation of preosteoblasts and precementoblasts into mature osteoblasts and cementoblasts and functions molecularly downstream from Runx 2. By inhibiting the Wnt/feration , 28, 53.β-catenin signalling pathway and of pathways of several members of BMP family. These pathways are essential for driving the process of commitment and subsequent differentiation of multipotential mesenchymal progenitor cells along the osteoblastic and cementoblastic lineages [Sclerostin is a glycoprotein encoded by the SOST gene, expressed by terminally differentiated cells within mineralized matrices, regulating osteoblast and cementoblast proliferation and differentiation, and ultimately playing an important role in bone formation, maturation, and remodelling , 58. Scllineages , 31, 59.2). Subsequently, PGE2 may mediate differentiation of cementoblasts and upregulate the expression of genes involved in mineral metabolism, including osteocalcin and alkaline phosphatase [Orthodontic load-generated strains on cementoblast precursors in the periodontal ligament induce Cox-2 mRNA expression and the synthesis of prostaglandin (PGEsphatase .β (TGF-β) superfamily, are found in high concentration in mineralized tissues. BMPs transduce intracellular signals through Smad-dependent molecular pathways and thorough Smad-independent molecular pathways, interacting with various transcription factors and regulating the expression of target genes [β-catenin signalling pathway, and depending on the tissue involved, these pathways may act synergistically or antagonistically [BMPs, members of the transforming growth factor-et genes . BMPs upet genes . The BMPstically . On the stically , 60, 61.β-catenin signalling by activating the Wnt/β-catenin inhibitors Dkk1 and sclerostin [BMP-2 promotes differentiation of cementoblast progenitor cells into a cementogenic lineage but has little mineralization-inducing effect on mature cementoblasts. Thus, any mineralization-promoting effect of BMP-2 is probably indirect through its interaction with progenitor cells, mediating the increase in the pool of mature cementoblasts , 62. On lerostin , 57.As previously mentioned, strategies to minimize external root resorption should include identification of systemic and local risk factors at the stage of treatment planning, limitation of treatment duration, the use of light intermittent forces, and biannual radiographic monitoring in order to detect any possible root resorption at the earliest stage . As someIt appears that if irregularities of root contour indicative of apical external root resorption are not detected within six to nine months of commencement of orthodontic treatment, then it would be unlikely that any significant apical external root resorption will occur, but if any apical external root resorption is observed within six to nine months from the beginning of orthodontic treatment, then there is a high risk of further root resorption , 65.If any apical external root resorption is detected, active treatment should be suspended for two to three months, hopefully to prevent further resorption and to allow some healing with cellular cementum. If further resorption is detected after active treatment has been resumed, the orthodontic treatment plan should be modified .