Multiple studies show that the soluble oligomers of Aβ42 are neurotoxic and proinflammatory, whereas the monomers and insoluble fibrils are relatively nontoxic. We show that Aβ42 aggregation is inhibited in vitro by oil palm phenolics (OPP), an aqueous extract from the oil palm tree (Elaeis guineensis). The data shows that OPP inhibits stacking of β-pleated sheets, which is essential for oligomerization. We demonstrate the inhibition of Aβ42 aggregation by (1) mass spectrometry; (2) Congo Red dye binding; (3) 2D-IR spectroscopy; (4) dynamic light scattering; (5) transmission electron microscopy; and (6) transgenic yeast rescue assay. In the yeast rescue assay, OPP significantly reduces the cytotoxicity of aggregating neuropeptides in yeast genetically engineered to overexpress these peptides. The data shows that OPP inhibits (1) the aggregation of Aβ into oligomers; (2) stacking of β-pleated sheets; and (3) fibrillar growth and coalescence. These inhibitory effects prevent the formation of neurotoxic oligomers and hold potential as a means to reduce neuroinflammation and neuronal death and thereby may play some role in the prevention or treatment of Alzheimer's disease.Alzheimer's disease is a severe neurodegenerative disease characterized by the aggregation of amyloid- The statistical analysis was made by performing one-way ANOVA for the 3 independent determinations. Significance of results was determined as β42. The Congo Red assay is based upon a spectral shift which occurs in the absorption of Congo Red at 2 different reference wavelengths when the Congo Red is bound to beta amyloid peptide monomers versus aggregates. The aggregation data in The Congo Red binding assay is one of the earliest methods developed to measure the aggregation of Aβ42 aggregation. We see that the concentration-inhibition relationship is somewhat linear between OPP concentrations of 1.0 and 7.0 μg/ml. Also it appears that maximum aggregation inhibition occurs at an OPP concentration of 7.0 μg/ml.As seen in β42 aggregation, the IC50 (50% inhibitory concentration) is 3.24 μg/ml OPP.Based on the above OPP inhibition of Aβ42 aggregation by OPP reveals both a dose-dependent and a time-dependent component.In β42 aggregate peaks representing dimers, trimers, tetramers, hexamers, and septamers. In β is seen at MW of 4,500 Da. The mass spectroscopy data clearly shows that the presence of OPP will inhibit the aggregation and polymerization of beta amyloid peptide. At a concentration of 10 μg/ml OPP, only the monomeric Aβ42 is present in solution.In μg/ml OPP, we see that only 2 species are found: the monomer and the dimer. Then from a concentration of 90 μg/ml up to 9 mg/ml, we see that only the monomeric form of the beta amyloid peptide is found.In β42 showing the frequency plots in the amide-I region, where the beta-sheets are characterized by the presence of two peaks centered near 1620 and 1680 cm−1, whose individual amide oscillators vibrate in-phase perpendicular or parallel to the β-strands, respectively. The splitting between these modes at this frequency is related to the size of the folded β-sheet. In a primarily beta-sheet protein, the corresponding cross-peaks give a characteristic Z-shape to the spectrum. Here the focus is primarily on the cross-peak centered at  =  cm−1 whose amplitude is indicative of the total amount of beta-sheet present in the sample.μg/ml OPP (c).Three 2D-IR spectra are shown in ω1, ω3] =  cm−1 (indicated by a red arrow) increases in amplitude over this period. This is indicative of an overall increase in beta-sheet content of the sample; we ascribe this increase in amplitude to growth of the beta-fibrils in the sample. Longer incubation times do not affect amplitude of the cross-peak (data not shown).The spectra in (a) and (b) show that the cross-peak at  and Aβ42 [42 amino acids in length]. Aβ42 peptide is known to be more fibrillogenic than Aβ40 so we have carried out these aggregation studies with Aβ42.Since the discovery that the beta amyloid peptide   . DynamicP (IC50) . The phahout OPP . Mass spwith OPP . The datμg/ml OPP, few small aggregates of Aβ42 can be visualized using transmission electron microscopy (TEM)  . Howeverg/ml OPP .μg/ml OPP rescues the growth of the β-amyloid-producing yeast from 20% (without OPP) to 40% (with OPP). For the Huntingtin-producing yeast, 10 μg/ml OPP rescues the yeast growth from 25% (without OPP) to 60% (with OPP). For the TDP-43-producing yeast, 10 μg/ml OPP rescues the yeast growth from 25% (without OPP) to 190% (with OPP). We see that there is no significant change of growth for the α-synuclein-producing yeast with or without OPP. These relative growth percentages compare the transgenic yeast growth to growth of control strains.In the transgenic yeast assay, the presence of 10 β40 and Aβ42 are the most common human isoforms of the peptide; Aβ40 is the more common isoform, but Aβ42 is more fibrillogenic, aggregates more quickly, and appears to be the more toxic isoform, especially in the dimer state. Aβ has little toxicity while in the monomeric state. Based upon neurotoxicity data in vitro, animal models, and clinical observations in patients with Alzheimer's disease, one therapeutic goal consists of inhibiting the formation of soluble Aβ dimers, thus nullifying the neurotoxic effect of these dimers and should be effective in preventing and/or treating Alzheimer's disease. Statistical studies on patients with AD have shown that the cortical levels of soluble Aβ correlate well with both the extent of synaptic loss and the severity of the clinical symptoms [β are the neurotoxic species which cause the cognitive losses of Alzheimer's disease [β (oAβ) have also been called Aβ-derived diffusible ligands (ADDLs). Recent research has shown that the oAβ/ADDLs play a key role in cognitive decline [Asymptoms , 105. Mu disease , 23–26.  decline , 28.β underlie the key phenotypic characteristics of Alzheimer's disease is supported by experimental data showing that these small soluble oligomers may (1) cause synaptic loss and decreased dendritic spine density; (2) cause hyperphosphorylation of tau proteins with resulting intraneuronal neurofibrillary tangles and collapse of the neuritic cytoskeleton; and (3) cause memory impairment and cognitive losses in the absence of amyloid plaques.The “amyloid hypothesis” that small soluble oligomers of A (Elaeis guineensis), have inhibitory effects on the process of aggregation and polymerization of beta amyloid peptide in vitro. This inhibition may cause the beta amyloid peptide to remain in the soluble monomeric state and facilitate the clearance of the peptide from the brain via the normal physiologic mechanisms. Several pharmaceutical companies are currently developing drugs which inhibit the aggregation and oligomerization of Aβ42. Preventing the formation of neurotoxic oligomers may represent a potential preventive or therapeutic strategy in the treatment of Alzheimer's disease.The experimental evidence presented in this paper shows that oil palm phenolics, derived from the palm oil plantβ42 aggregation may lead to the development of a potential drug for the prevention and/or treatment of Alzheimer's disease. Our next step consists in identifying, isolating, and purifying that component(s) of the oil palm phenolics which has these inhibitory and antifibrillogenic effects on beta amyloid peptide.Thus, the inhibitory effects of OPP on Aβ-amyloid peptide in vitro and the inhibitory effects of OPP on this aggregation. The study led to the following experimental results:β-amyloid monomers from aggregating into dimers, trimers, or larger aggregates as shown by the observed reduction in aggregate size as measured by Congo Red binding, mass spectrometry, dynamic light scattering, 2D-IR spectroscopy, and transmission electron microscopy.Oil palm phenolics inhibit β42 oligomerization as observed by dynamic light scattering with a shift of the aggregation curves to the right.Oil palm phenolics prolong the lag phase in the nucleation-elongation process of AOil palm phenolics inhibit the folding of the beta-pleated sheet as observed in the 2D infrared spectroscopy.μg/ml (IC50) by Congo Red dye binding.Fifty percent (50%) of the maximum aggregate size is observed at an OPP concentration of 3.24 μg/ml, only monomers of Aβ42 are observed by mass spectrometry. No dimers, trimers, or higher oligomers are observed at this concentration or higher.At an OPP concentration greater than or equal to 90 μg/ml in transgenic yeast which overexpresses these peptides.The cytotoxicity of aggregated neuropeptides is greatly reduced by OPP at a concentration of 10 This is a study of the aggregation of β42 oligomers are neurotoxic and cause extensive pathologic changes in neurons, decrease dendritic spine density, and cause depression of long-term potentiation in neurons and enhancement of long-term depression. Therefore, the properties of the oil palm phenolics to inhibit the formation of oligomers may hold promise for the medical treatment and/or prevention of Alzheimer's disease.The current scientific literature shows that the soluble A


Neuroblastoma (NB) is a tumor of the sympathoadrenal system arising in children under 15 years of age. In Germany, NB accounts for 7% of childhood cancer cases, but 11% of cancer deaths. It originates from highly migratory progenitor cells that leave the dorsal neural tube and contribute neurons and glial cells to sympathetic ganglia, and chromaffin and supportive cells to the adrenal medulla and paraganglia. Clinically, histologically and molecularly, NBs present as extremely heterogeneous, ranging from very good to very poor prognosis. The etiology of NB still remains unclear and needs to be elucidated, however, aberrant auto- and paracrine embryonic cell communications seem to be likely candidates to initiate or facilitate the emergence, progression and regression of NB. The wingless-type MMTV integration site (WNT) family of proteins represents an evolutionary highly conserved signaling system that orchestrates embryogenesis. At least 19 ligands in the human, numerous receptors and co-receptors are known, which control not only proliferation, but also cell polarity, migration and differentiation. Here we seek to interconnect aspects of WNT signaling with sympathoadrenal and paraganglionic development to define new WNT signaling cues in the etiology and progression of NB. Neuroblastoma (NB) is a tumor of the sympathetic nervous system that arises in children under 15 years of age with a frequency of 1:5700. In Germany, NB accounts for 7% of childhood cancer cases, but is responsible for approx. 11% of cancer deaths in children , 3). It Classically, NB are staged into five groups 1–4, and 4S) according to localization, lymph node involvement, and metastasis formation , 8. Curr, and 4S MYCN is the strongest indicator for highly malignant and therapy-resistant NB. Despite initially successful therapy, these patients frequently suffer from relapse, and die because of metastasis formation and resistance to chemotherapy . Howeveiewed by –20.Glomus caroticum and the Glomera aortica, which are located along the glossopharyngeal and vagal nerves, and represent sensory organs . They should not be mixed up with the sympathoadrenal–paraganglionic system, despite their common origin from the neural crest , 2928, 2It still is not completely understood how the fate of NC cells is determined. Some of them may already be pre-determined when they leave the neural tube, however, differentiation is also regulated by environmental signals the cells receive during their migratory route (recently reviewed by ). There It has still remained elusive how the different NC cell populations are determined. Most likely, environmental interactions both during migration and in the target organ are of great importance. Exchange of sympathetic and vagal NC has revealed that the emigrating cells are capable to adapt to their new environment, and can produce both cholinergic parasympathetic and adrenergic sympathetic neurons depending on the environment , 31, 40.Beside chromaffin cells, in the adrenal medulla there are additional cell populations of sympathoadrenal neural crest origin. Even though their function has not been finally elucidated yet, these are multipolar sympathetic neurons that may regulate blood flow in the adrenal medulla, ‘sustentacular cells’ with glial characteristics, and small cells with a mixed adrenergic/noradrenergic character, which might be chromaffin stem cells . So far WNT signaling is a crucial and highly conserved pathway in the development of vertebrates as a whole, and specifically also for the development of the peripheral nervous system. In the human, 19 WNT ligands are known so far, which are secreted in a distinct temporal and spatial order, thereby regulating cell proliferation, survival, migration, polarity, and the fate of stem cells , 48. The2+ pathway. WNT1, -2, -3 and -8 are supposed to act in the β-catenin-dependent pathway(s), whereas WNT4, -5 and -11 are regarded as activators of the β-catenin-independent pathways. However, WNT ligands were also shown to act via interconnected intracellular networks, depending on the cellular context [WNT ligands and receptors address several downstream signaling cascades, which are commonly divided into the β-catenin-dependent  and the two β-catenin-independent pathways, the planar-cell-polarity (PCP) and WNT–Ca context –52. In a context –55. Inst context , 56).Another obstacle in understanding WNT signaling is the fact that some ligands and receptors can substitute for each other. This may provide a safeguard of the system or serve as a potent tool to ensure a precise temporal and spatial regulation of WNT-driven activations during development, by addressing different promotor structures for the same (or similar) cellular behavior. However, deregulation of WNT signaling is the cause of various types of cancer In vertebrate embryos, the formation of the NC consists of at least two crucial steps. The first one is the induction of NC cells during neurulation in the neural plate. This initiation requires FGF, retinoic acid and WNT signals (for review see ). The se2+-dependent WNT pathway [After they have reached the postmitotic stage, sympathetic neurons start expanding their axons toward the innervation target. Molecules involved in this step are hepatocyte growth factor, Artemin, NT3, and nerve growth factor (NGF). Notably, TRK-A, the receptor for NGF, is a favorable prognostic marker in NB , 74. How. How4. F pathway . These f2+ signaling . Our exiewed in .Table 1EVANGL2 and PRICKLE1 correlates with low-risk NB and reduced levels of active β-catenin [VANGL2 and PRICKLE1 are key players of the  PCP-pathway. Inhibition of ROCK1/2, central molecules of PCP, increases PRICKLE1 expression and inhibits β-catenin pathway activity. This suggests a direct inhibition of β-catenin-dependent WNT signaling by PCP via PRICKLE1. Another recent publication focused on transcriptome analyses and computational interaction studies in NB, melanoma and colorectal carcinoma, and provided data that correlate high WNT3A, WNT5A, APC, or FZD10 expression with longer event-free survival, while high WNT3 or FZD1 indicates less favorable outcome [MYCN activity at various levels, and provided a gene signature based on the WNT pathway, which may improve risk stratification for NB patients.So far, WNT signaling in NB has been a neglected topic, and only few studies have provided evidence for the impact of WNTs in NB, and demonstrated that the dissection of WNT signaling into the so-called canonical and non-canonical pathways may not properly reflect the heterogeneity of the lesions Fig. . WNT pat-catenin . Both VA outcome . This grMYCN non-amplified cell lines [MYCN-amplified and MYCN-non-amplified cell lines  efflux-transporter that confers drug resistance to tumor cells . KnockdoMYCN-amplified high-risk tumors, differentiation therapy often fails. Very recent expression analyses show that DKK1 and FZD7 are inversely regulated by MYCN and RA, and may be part of the RA-induced differentiation signaling cascade [Retinoids (RA) are standard drugs used for differentiation therapy in NB –92. Howe cascade . Failure cascade . High HI cascade , 95, and cascade . Inhibit cascade , 98. ThiMYCN-amplified tumors, and positive correlation with adverse outcome [The functional homologues LGR4 and LGR5 are G-protein-coupled receptors that bind R-spondins, and thereby amplify β-catenin-dependent WNT signaling   outcome . Our expLGR4/-5 expression is also of interest when considering developmental aspects. As mentioned above, migrating sympathetic neuroblasts require BMP from the dorsal aorta to differentiate into postganglionic neurons during formation of sympathetic ganglia , 43. MorALK, and especially in the RAS/MAPK-signaling pathway [NBs frequently present with chromosomal aberrations. Thereby, loss of genetic material can be observed at chromosomes 1p and 11q, whereas gain of genes can be found at chromosomes 11p and 17q , 14. A r pathway . However pathway . Interes pathway , 105. HoMYCN, most likely via miRNA-mediated mRNA degradation [Furthermore , there may be a shift in WNT signaling from β-catenin-independent signaling ligands (WNT4A and WNT11), which are located in loss-prone regions to β-catenin-dependent signaling (represented by WNT3) on the gain region. This may be enhanced by increased expression of LGR4 as discussed above  radation –112. Expradation . HoweverIn addition to WNT signaling, there are other genes on the affected chromosomal regions linked to the development of the sympathoadrenal system Table . FailureAs discussed above, WNT signaling seems to offer multiple targets for new therapeutic strategies. Thereby, both β-catenin-dependent and independent pathways may be suitable. Very recently, FZD2 has been shown to interact with WNT5A and WNT3A, thereby activating ‘canonical’ and ‘non-canonical’ WNT signaling in NB cells . KnockdoWNT/β-catenin signaling is also the focus of drug optimization approaches. Tankyrase1 (TNKS1) is known to phosphorylate Axin1/2, thereby regulating the β-catenin destruction complex. Inhibition of TNKS1 with XAV939 stabilizes this complex and increases β-catenin destruction, which prevents WNT/β-catenin-signaling , 116. ThCytotoxic therapy might also be improved by combining chemotherapy with LiCl, an activator of the WNT/β-catenin pathway, as shown in cell culture experiments . Recentl2+ pathways are available. Additionally, β-catenin-dependent WNT pathways were shown to play a pivotal role in NB expansion and progression. Analyses of chromosomal aberrations reveal a possible shift from β-catenin-independent to classical WNT–β-catenin pathways with a possible key role for the loss of FZD4 expression. Both β-catenin-dependent and independent pathways facilitate a broad spectrum of cell–cell interactions, and it is highly likely that there are mechanisms that link the genesis of NB to early developmental processes. The heterogeneity of NB and the complexity WNT signaling are clinically and scientifically challenging. Considering the similarities between the embryonic development of the sympathoadrenal–paraganglionic system and the biology of NB, progression in WNT signaling research in one field may be transferred to the other, which may finally result in new and improved therapeutic strategies for the benefit of the young patients.According to our current knowledge, WNT signaling only seems to be indirectly involved during early development of the sympathoadrenal system. However, at the stage of direct target innervation, WNT5A and ROR signaling is indispensable for terminal differentiation of adrenergic neurons. Regarding NB, several groups have published (in part conflicting) results, which once more depict the immense heterogeneity of this sympathoadrenal tumor. Nevertheless, the data presented so far strongly suggest a critical role for WNT signaling in NB. Our expression data Table  support


APC, AXIN2, CTNNB1, and ZNRF3 in ACCs. Mutations in APC, CTNNB1, ZNRF3 and homozygous deletions in ZNRF3 were observed in 3.8% (2/52), 11.5% (6/52), 1.9% (1/52) and 17.3% (9/52) of the cohort respectively. Novel interstitial deletions in CTNNB1 spanning intron 1 to exon 3/intron 3 were also found in 7.7% (4/52) of the tumours. All the observed alterations were mutually exclusive. Nuclear accumulation of β-Catenin, increased expression of Cyclin D1 and significantly higher expression of AXIN2 (p = 0.0039), ZNRF3 (p = 0.0032) and LEF1(p = 0.0090) observed in the tumours harbouring the deletion in comparison to tumours without CTNNB1 mutation demonstrates that the truncated β-Catenin is functionally active and erroneously activates the downstream targets. Significantly lower overall survival rate in patients with tumours harbouring alterations in APC/CTNNB1/ZNRF3 in comparison to those without mutation was observed. In conclusion, the discovery of novel large deletions in addition to the point mutations in CTNNB1 infers that activation of Wnt/β-Catenin pathway via alterations in CTNNB1 occurs frequently in ACCs. We also confirm that alterations in Wnt/β-Catenin signaling pathway members have a negative effect on overall survival of patients.The Wnt/β-Catenin signaling pathway is one of the most frequently altered pathways in adrenocortical carcinomas (ACCs). The aim of this study was to investigate the status of Wnt/β-Catenin signaling pathway by analyzing the expression level of β-Catenin and the mutational status of  In the absence of Wnt signaling, β-Catenin bound by the destruction complex  is phosphorylated by CK1 and GSK3β on its serine/threonine residues. This leads to ubiquitin-mediated proteolysis of β-Catenin. However, in the presence of Wnt signaling, Wnt ligands bind to their FZD/LRP receptors leading to recruitment of destruction complex to the plasma membrane followed by phosphorylation of GSK3β. This interferes with the destruction complex’s activity and promotes stabilization and accumulation of newly synthesized β-Catenin with subsequent transcription activation or inhibition of the downstream target genes2. Recently R-spondin and ZNRF3/ RNF43 module has been recognized as an upstream regulator of Wnt/ β-Catenin signaling pathway3. ZNRF3/ RNF43 are members of E3 ubiquitin ligase family and have been found to reduce the Wnt signaling by ubiquitination-mediated degradation of Wnt receptors, Frizzled. In the presence of R-spondin , however, ZNRF3/RNF43 are cleared from the membrane which eventually leads to accumulation of Wnt receptors and increased Wnt signaling.The Wnt/β-Catenin signaling pathway is vital for adrenal development, differentiation, and cell renewal6. The N-terminal domain harbours serine/threonine residues which serve as GSK3β and CK1-mediated phosphorylation sites, and are recognized by β-TrCP ubiquitin ligase9. Mutations in exon 3 of the CTNNB1 gene (encoding β-Catenin) causing loss of serine/threonine residues are the most frequent mechanism of an erroneously activated Wnt/β-Catenin signaling pathway12. Sporadic cases of deletions and insertions in exon 3 and deletion of the entire exon 3 have also been observed13.Structurally, β-Catenin largely consists of three domains; the N-terminal domain, central armadillo repeats and the C-terminal domain. The central region (residues 141–664) consists of 12 armadillo repeats and is the most conserved region of β-Catenin harbouring binding sites for interaction with other proteins such as APC, Axin, and TCFCTNNB1 are known to occur in different types of adrenocortical adenomas and cancers15. Adrenocortical carcinoma (ACC) is a rare type of malignancy with an incidence of 1–2 per million16. CTNNB1 mutations have been observed in approximately 10–15% of ACCs20, although aberrant activation of Wnt/β-Catenin signaling is observed in a much higher proportion of ACC tumours, most of them are not caused by known CTNNB1 mutations21. Recent pan-genomic studies of ACCs have shown that alterations in other members of Wnt/β-Catenin signaling pathway such as ZNRF3 and APC could also lead to activation of the pathway19. Furthermore, benign adrenal tumours with aberrant β-Catenin nuclear accumulation have been associated with a larger size and malignant adrenal tumours with aberrant β-Catenin nuclear accumulation defined poor prognosis21.Aberrations in Wnt/β-Catenin signaling through activating mutations of APC, AXIN2, CTNNB1 and ZNRF3 and expression of β-Catenin in a large cohort of ACCs. We also describe novel interstitial deletions in CTNNB1 and their functional impact in ACCs.Here we have analyzed the mutation status of APC, AXIN2, CTNNB1, and ZNRF3. Mutations in APC, CTNNB1, and ZNRF3 genes were found in two, six and one sample respectively whereas none of the analyzed samples harboured any mutations in AXIN2. One of the APC mutations was germline, c.4666_4667insA (p.T1556NfsTer3), and the other was a somatic mutation, c.4391_4394del (p.E1464GfsTer8) , c.104T > A (p.I35N), c.136C > A (p.S45Y), c.109C > G (p.S37C), c.105G > A (p.G34R) and c.134C > T (p.S45F) were found in 6 individual tumours , was found in ZNRF3  to that of European subpopulations22.We have screened 61 tumours originating from 52 patients for mutations in members of Wnt/β-catenin signaling pathway; CTNNB1 was performed on cDNA of all 61 ACC tumours to identify deletions spanning exon 3. Visualization of the products through gel electrophoresis revealed presence of shorter bands in four tumours  of approximately 630 bp in size along with a 920 bp apparently wild-type product ; we performed whole genome sequencing in one tumour harbouring the deletion (ACC40). We found a deletion of 6407 bp (g.41218317-g.41224723del) spanning over intron 1 to exon 3  deletion on the protein product, β-Catenin, we performed Western blot analysis on the tumour protein lysates, which demonstrated expression of wildtype as well as truncated protein in samples harbouring the ∆(2 + 3) deletion  deletion in comparison to those without mutations in analyzed genes (ACC wildtype (n = 39) vs ∆(2 + 3) deletion (n = 4), p = 0.0039 and p = 0.0090 respectively) and was comparable to the tumours harbouring CTNNB1 missense mutations (ACC wildtype (n = 39) vs CTNNB1 mutants (n = 5), p = 0.0211 and p = 0.0451 respectively; Fig. CTNNB1 mutations  in the samples with the deletion in comparison to analyzed tumours without ion Fig. . mRNA exons Fig. . A trendZNRF3 locus (q < 0.01). Homozygous deletions at ZNRF3 locus were observed in 9/52 tumours. Analysis of SNP array data from recurrent tumours available for 4/9 cases with ZNRF3 deletion in the primary tumour showed presence of ZNRF3 deletion in all four recurrent tumours. The deletions found in ZNRF3 were mutually exclusive to APC, CTNNB1 and ZNRF3 mutations. Samples with ZNRF3 deletions showed significantly lower expression of ZNRF3 (p = 0.0001) and higher expression of AXIN2 (p = 0.0274) and LEF1 (0.0081) in comparison to the samples devoid of mutations in investigated genes , 15 samples harboured loss, nine harboured gain, 11 harboured copy neutral loss of heterozygosity (cnLOH), five harboured loss with allelic imbalance whereas 11 were diploid at the CTNNB1 locus 3p22.1  deletion, two harboured gain and one harboured cnLOH  showed significant deletions occurring at ZNRF3 alterations (8 deletions and 1 mutation), only three samples showed expression of the active β-Catenin.Active β-Catenin expression status was assessed by Western blot analysis for all the available primary tumours (n = 50). Active β-Catenin expression was detectable in 31/50 analyzed tumours , (2) with CTNNB1 mutation but without nuclear expression (n = 1), (3) with wildtype CTNNB1 and without nuclear expression (n = 23) and (4) with wildtype CTNNB1 and nuclear expression (n = 16) , this revealed cytoplasmic expression in 45 tumours and nuclear expression in 25 tumours  Figs  and S11.Correlative analyses of nuclear accumulation and active β-Catenin expression were performed on 44 primary tumours analyzed by both methods  novel interstitial deletion, ∆(2 + 3) deletion, in CTNNB1 leading to expression of a truncated protein. To our knowledge, such interstitial deletions leading to skipping of exon 2 and 3 have not been reported in adrenal tumours. Deletion of exon 2 and 3 in CTNNB1 was previously observed in one melanoma cell line23 and in one malignant melanoma24. Such deletion of a whole exon could occur due to several phenomena such as interstitial deletions at the genomic level, alternative splicing or other post-translational processing. However, in both of the studies the mutation was observed in and limited to cDNA derived from the specimens and hence was not conclusive regarding the cause of the deletion.Mutations and deletions in genes involved in the Wnt/β-Catenin signaling pathway are observed in approximately 40% of ACCs and mutations in CTNNB1. Interestingly, deletions occurred at similar regions in all four tumours leading to deletion of exon 2 and either whole (n = 1/4) or fractions of exon 3 (n = 3/4). Despite deletion of exon 2 carrying the transcription start site, and complete exclusion of exon 3, expression of an in-frame truncated protein was observed in all four tumours. This implicates that for the production of the in-frame protein product, the transcription and translation machinery skips the remainder of exon 3 and starts translation from another in-frame start codon. Generally, an exon can harbour an exonic splicing enhancer (ESE) or an exonic splicing silencer (ESS) element25. ESE is required for the efficient splicing and inclusion of the exon it resides in whereas ESS is required for skipping. Alterations in ESE elements are known to cause either inefficient splicing, leading to partial or complete exclusion of the exon27. This and several other types of nonsense-mediated alternative splicing mechanisms could explain the complete exclusion of exon 3 in these tumours27.Whole genome sequencing allowed us to map interstitial deletions spanning intron 1 to exon 3/intron3 of 28. Deletions or mutations in this region cause stabilization and accumulation of β-Catenin23. Constitutively active β-Catenin occurring via deletion of exon 3 alone was shown to induce adrenal hyperplasia in female mice, which had advanced to cancer in some cases29. N-terminal deleted β-Catenin with intact highly conserved armadillo repeats important for its structural and functional activity30, accumulates in the cytoplasm and nucleus31 and mimics activated Wnt signaling pathway in Xenopus and Drosophila32. The observed ∆(2 + 3) deletion in β-Catenin in our study causes a removal of first 87 amino acids given that the first in-frame start site in exon 4 is utilized. Such N-terminally deleted β-Catenin, lacking the first 86 and 89 amino acids (∆N86 and ∆N89 β-Catenin) was shown to cause increased LEF1 mediated transcription in vitro and increased proliferation, apoptosis and decreased migration in mouse intestinal epithelial cells33. ∆N89 β-Catenin expression in mouse mammary glands was shown to cause development of aggressive adenocarcinomas in 100% of the studied female mice34. These observations provide an excellent model for ∆(2 + 3) deletion effects.β-Catenin turnover relies on the presence of its serine/threonine rich N-terminal region that becomes phosphorylated by GSK3β and CK135. Activation of Wnt/β-Catenin signaling increases the expression of ZNRF33, LEF1, Cyclin D136 and AXIN2, which are direct targets of β-Catenin38. Similar to AXIN2, ZNRF3, a newly added member of the Wnt/β-Catenin signaling pathway, is a negative regulator of the pathway3. ZNRF3 is also the most frequently deleted gene in ACCs19 and is reported to be upregulated in colorectal tumours exhibiting activated Wnt/β-Catenin signaling pathway3. Our findings of high expression of Cyclin D1 and significantly higher expression of AXIN2, LEF1 and ZNRF3 mRNA in tumours harbouring the ∆(2 + 3) deletion at a similar level to those with hot spot mutations suggest the activating nature of these deleterious mutations in ACCs.Immunohistochemical analysis of the tumours with mutant β-Catenin showed nuclear and cytoplasmic accumulation of β-Catenin in all but one tumour with the ∆(2 + 3) deletion. An absence of nuclear accumulation of β-Catenin in adrenocortical tumours harbouring activating β-Catenin mutations has been observed before21. Association of CTNNB1 mutation with low survival observed in our cohort confirms and validates the previous observations. In our study, the association was even more evident in the group of patients with tumour harbouring alterations in APC/CTNNB1/ZNRF3.Mutations in members of Wnt/β-Catenin signaling pathway and nuclear accumulation of β-Catenin are correlated with poor survivalCTNNB1 missense mutation and ZNRF3 deletion) and accumulation of β-Catenin do not always occur together35. In addition to such observation in our study, we also observed that tumour samples with the accumulation of nuclear β-Catenin did not always show accumulation of active β-Catenin and vice versa. Tumour heterogeneity and complexity of malignant tumours like a culmination of whole genome hypo- or hyperploidy, accumulation of mutations and alterations in methylation, RNA expression and miRNA expression which is also observed in ACCs might lead to different phenotypic observations in tumours than expected by just a presence of the single mutations.While aberrations in Wnt/β-Catenin signaling pathway and nuclear accumulation of β-Catenin are observed with similar frequency (40%) in ACCs, the presence of aberrancy  were operated for adrenocortical cancer (ACC), with adrenalectomy. Amongst 52, five cases were childhood ACC  tissue samples. All biologic specimens were obtained from Uppsala Biobank, Endocrine tumour collection . The study was approved by the regional ethical review board in Uppsala . Ethical approval and informed consent from all the included patients (from guardians if younger than 18) were obtained prior to the study.Tumours were cryosectioned and hematoxylin-eosin stained to assure >80% tumour content. When necessary, after histological analysis, tumours were macro-dissected, to achieve maximum tumour cell content. DNA and RNA were prepared from cryosectioned tumour samples and matching normal tissue using Allprep DNA and RNA kit . Blood DNA was prepared using Blood and Tissue kit . Quality controlled RNA was used to prepare cDNA using RevertAid First strand cDNA Synthesis kit .APC and AXIN2 were described before40. Obtained PCR products were visualized using 1.5% Agarose gel with SYBR green. The PCR products were gel excised when necessary and purified using PureLink Quick Gel Extraction Kit . PCR products were sequenced using automated Sanger sequencing at Beckman Coulter Genomics, UK. The sequence chromatograms were analyzed using Codoncode Aligner 3.7.1  using NM_001904.3 as a reference sequence. Beta-actin was used as a reference gene for qPCR analysis. SsoAdvanced SYBR Green Supermix  was used to perform all the qPCRs on a CFX96 Real Time system . The samples were run in triplicates and the ∆∆CT method was used to determine the relative expression levels. Log-transformed values were used for graphical representation.Specific primers for DNA and cDNA were used to amplify the regions of interest  at the Science For Life core facility . The raw data were processed with Illumina BeadStudio, and further processed and analyzed using Nexus copy number variation 7.5 software . Allele-Specific Copy number Analysis of Tumours (ASCAT) analysis was performed on the SNP array data. The samples were manually re-centered using ASCAT data. Copy number variation analysis was performed using logR ratio and B allele frequency (BAF). For the analysis of frequent deletions occurring in the cohort, the SNP array data was first processed using circular binary segmentation algorithm and were further analyzed using GISTIC tool2O2) followed by (4) heat treatment using citric buffer for antigen retrieval (5) sections were incubated with normal horse serum , (6) anti- β-Catenin  and (7) secondary antibody  diluted in BSA-PBS buffer . Chromogenic detection of protein using (8) ABC and (9) DAB  and (10) nuclear staining using hematoxylin followed. The slides were washed with PBS between each step mentioned above. The sections were then (11) dehydrated by treating them with 70% ethanol, 90% ethanol, 99.9% ethanol and xylene. For frozen tissue immunohistochemistry, 5 μm thick cryosections were generated and (1) fixed in acetone, (2) treated with hydrogen peroxide (H2O2) and (3) blocked with avidin-biotin. Steps 5, 6, 7, 8, 9 and 10 mentioned above were followed for staining of cryosections.Immunohistochemistry was performed on 4 μm thick FFPE tissue sections: (1) The slides were dried at 60 °C before use (2) sections were treated with xylene, 99.9% ethanol, 90% ethanol, 70% ethanol and dH2O in a series, for paraffin removal and rehydration of the tissue sections (3) sections were treated with hydrogen peroxide  and “positive” for nuclear staining. For positive cases, the nuclear and cytoplasmic staining intensity was scored manually ascending from 1+ to 5+, independently by two investigators blinded to the mutation status of the investigated genes. Scoring for heterogeneous cases was based on the positively stained sections solely. For graphical representation, the samples with 1+ to 2+ were grouped as ‘low’ and samples with 3+ to 5+ were grouped as ‘high’.47. Following read alignment, duplicate reads were excluded and base quality score recalibration was performed using GATK v. 3.350. Insertions, deletions and other structural variants located on chromosome 3 were called on the generated BAM file with Pindel v.0.2.451, using the standard settings and an average insert size of 250 bases. Possible deletion/insertion overlapping the CTNNB1 locus was manually inspected.Extracted DNA was subjected to whole genome sequencing at the SNP&Seq Technology Platform in Uppsala on an Illumina HiSeq. 2500/HiSeqX. The number of generated reads was 667,706,189, out of which 650,970,282 were aligned to the reference resulting in a mean autosomal coverage of 29.67x. The generated reads were aligned to the reference genome (human_g1k_v37) using BWA v. 0.7.1252 and probed for β-Catenin , active β-Catenin , Cyclin D1  and Actin . The expression levels of the protein were normalized using a reference (ACTB) and were quantified using Image Lab software .Samples were lysed using cytobuster  supplemented with protease inhibitor cocktail . Obtained protein lysates were denatured at 95 °C with Laemmli buffer and β-mercaptoethanol for 10 minutes. The heat-treated samples were subjected to SDS-PAGE and transferred to a PVDF membrane. Western blotting was performed on the PVDF membrane as described beforeThe Mann-Whitney U test was used for quantitative analysis of Cyclin D1 western blot. Kruskal-Wallis test with correction for multiple comparisons by controlling False discovery rate using the two-stage step-up method of Benjamini, Krieger, and Yekutieli was performed for statistical analysis of AXIN2, Cyclin D1, LEF1 and ZNRF3 mRNA expression, utilizing GraphPad Prism 7 . Cox proportional hazards regression method was used to determine univariate and multivariate hazard ratios using SPSS software. For survival analysis, the data were censored if the patients were still alive at last follow up. Survival analyses were made using survival curves created by the Kaplan-Meier estimate with the Log-rank test. Statistical significance was determined to be achieved when p < 0.05. The Chi-square tests were used for comparison of nominal data.Supplementary Information


Altered metabolism is one of the hallmarks of cancers. Deregulation of ribose-5-phosphate isomerase A (RPIA) in the pentose phosphate pathway (PPP) is known to promote tumorigenesis in liver, lung, and breast tissues. Yet, the molecular mechanism of RPIA-mediated colorectal cancer (CRC) is unknown. Our study demonstrates a noncanonical function of RPIA in CRC. Data from the mRNAs of 80 patients’ CRC tissues and paired nontumor tissues and protein levels, as well as a CRC tissue array, indicate RPIA is significantly elevated in CRC. RPIA modulates cell proliferation and oncogenicity via activation of β-catenin in colon cancer cell lines. Unlike its role in PPP in which RPIA functions within the cytosol, RPIA enters the nucleus to form a complex with the adenomatous polyposis coli (APC) and β-catenin. This association protects β-catenin by preventing its phosphorylation, ubiquitination, and subsequent degradation. The C-terminus of RPIA (amino acids 290 to 311), a region distinct from its enzymatic domain, is necessary for RPIA-mediated tumorigenesis. Consistent with results in vitro, RPIA increases the expression of β-catenin and its target genes, and induces tumorigenesis in gut-specific promotor-carrying RPIA transgenic zebrafish. Together, we demonstrate a novel function of RPIA in CRC formation in which RPIA enters the nucleus and stabilizes β-catenin activity and suggests that RPIA might be a biomarker for targeted therapy and prognosis. The pentose phosphate pathway generates NADPH, pentose, and ribose-5-phosphate by RPIA for nucleotide synthesis. Deregulation of RPIA is known to promote tumorigenesis in liver, lung, and breast tissues; however, the molecular mechanism of RPIA-mediated CRC is unknown. Here, we demonstrate a role of RPIA in CRC formation distinct from its role in these other tissues. We showed that RPIA is significantly elevated in CRC. RPIA increased cell proliferation and oncogenicity via activation of β-catenin, with RPIA entering the nucleus to form a complex with APC and β-catenin. Further investigation suggested that RPIA protects β-catenin by preventing its phosphorylation, ubiquitination, and subsequent degradation. In addition, the C-terminus of RPIA (amino acids 290 to 311), a portion of the protein not previously characterized, is necessary for RPIA-mediated tumorigenesis. Finally, we observed that transgenic expression of RPIA increases the expression of β-catenin and its target genes and induces tumorigenesis. Our findings suggest that RPIA can enter the nucleus and associate with APC/β-catenin, and suggest precise treatment of human CRC by targeting its nonenzymatic domain. Cyclin D1 (CCND1) and Cyclin E2 (CCNE2). Abnormal activation of the β-catenin signaling pathway can lead to increased cell proliferation and immortalization [Colorectal cancer (CRC) is one of the most common forms of cancers and results in more than 600,000 deaths annually –3. Mutatlization –19. For lization . HoweverG12D, an activated form of Ras [The pentose phosphate pathway (PPP) is critical for cancer cell survival and proliferation ,22. Ribom of Ras . Our prem of Ras . Studiesccne1. Our work uncovers a new role of RPIA and provides a molecular mechanism of RPIA-mediated β-catenin stabilization and activation necessary for colon cancer formation.Here, we report that in CRC tissue RPIA is significantly up-regulated, and it is expressed at multiple stages of tumorigenesis, including early stages. It directly interacts with β-catenin and APC to activate target genes downstream of β-catenin that are important for carcinogenesis. High levels of RPIA expression stabilize β-catenin levels by preventing phosphorylation and ubiquitination of β-catenin. Transgenic zebrafish overexpressing RPIA under the control of a gut-specific promoter exhibited enhanced β-catenin expression and elevated mRNA levels of the colon cancer marker gene To assess the role of RPIA in the progression of CRC, we measured RPIA protein levels using immunohistochemistry (IHC) with tissue arrays from stage I through IVB and metastatic colon cancer. The RPIA immunoreactive score (IRS) was calculated by multiplying the staining intensity by the proportion of positive cells  S1A FigS1A Fig. To examine the effects of RPIA overexpression on cellular proliferation, two colon cancer cell lines, HCT116 and SW480, were used. SW480 is a human colon cancer cell line with APC C-terminal truncation at 1338, but the β-catenin binding region is retained; HCT116 is a highly metastatic cell line with WT APC and both an S45 mutation and the WT allele for β-catenin, but most of the β-catenin protein comes from the mutant allele. WST-1 assays examine metabolic activity that represent the viability of the cell. We tested three small interfering RNAs (siRNAs) , which wAberrant β-catenin accumulation is a major cause of uncontrollable proliferation in colon cancer cells ,27. β-caCTNNB1 gene) mRNA levels. Conversely, RPIA overexpression increased β-catenin protein expression levels in both HCT116  was used to determine the half-life of β-catenin in cell lines with either reduced or elevated levels of RPIA. Reduction of RPIA by knockdown decreased the β-catenin protein half-life from 3 to 1.1 h in HCT116 cells  and fromser9), which does not have the ability to phosphorylate β-catenin, were further examined. Because phosphorylated GSK3βser9 was elevated upon overexpression of RPIA in both HCT116 and SW480 cell lines and there is no difference in the nucleus GSK3β, we suggest the RPIA modulate GSK3β only in the cytoplasm , mice (Mus musculus), and zebrafish (Danio rerio) (The RPIA protein sequence is conserved among humans (o rerio) . To deteo rerio) . The WT o rerio) . We notio rerio) . These do rerio) . Moreoveo rerio) . Furthero rerio) . These rifabp). In particular, we analyzed the histopathology of the intestinal bulb (IB), middle intestine (MI), and posterior intestine (PI) collected from non-transgenic and transgenic (ifabp:RPIA) fish of different ages. Increased nuclear-to-cytoplasmic ratio, nuclear atypia, and moderately differentiated adenocarcinoma were observed in 3- and 5-month-old Tg (ifabp:RPIA) fish relative to the WT controls  fish, and the expression levels of β-catenin target genes were found to be positively correlated with RPIA expression levels (ccne1 is required for cell cycle/proliferation and tumor growth in CRC [ccne1 were elevated in 3-month-old Tg (ifabp:RPIA) fish, especially in the PI, which had dramatically increased RPIA expression. In addition to 3-month-old fish, we also analyzed β-catenin target genes in 5-month-old fish (ifabp:RPIA) fish, the β-catenin target genes were up-regulated more significantly in the PI than in the IB  of the NHRI and were in accordance with International Association for the Study of Pain guidelines (protocol number: NHRI-IACUC-104157-A). Taiwan Zebrafish Core Facility (TZCF) at NHRI or TZeNH is a government-funded core facility, and since 2015, the TZeNH has been AAALAC accredited.The CRC cell lines HCT116 and SW480 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin, and 100 μg/ml of streptomycin and incubated at 37°C and 5% carbon dioxide. DNA typing of the cell lines was verified by Mission Biotech  using a Promega GenePrint 10 System.Transient transfection of siRNA was performed using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s manual. Three individual RPIA siRNAs  and si-NC were purchased from Invitrogen.pcDNA3.0-RPIA, RPIA-ΔA, RPIA-ΔB, RPIA-Δ(A+B), RPIA-ΔC, and RPIA-ΔD were constructed by subcloning full-length or truncated RPIA cDNAs into a pcDNA 3.0 expression vector. Truncated RPIA products were amplified by specific primer sets: RPIA-ΔA (nt 1–522 deletion) (forward) 5ʹ ATAGAATTCATGGGCGGAGGCTG CCTGAC 3ʹ and (reverse) 5ʹ AGACTCGAGCTTGCAGGGTCAACAGAAAGGCT 3′; RPIA-ΔB (nt 523–567 deletion) (forward) 5ʹ ATAAGTCGCTTCATCGTGATCGCT 3ʹ and (reverse) 5ʹ AGAACCCTTGATGAGATTGAGATCA G 3′; RPIA-Δ(A+B) (nt 1–567 deletion) (forward) 5ʹ ATAGAATTCATGAGTCGCTTCATCGTGATCGCT 3ʹ and (reverse) 5ʹ AGACTCGAGCTTGCA GGGTCAACAGAAAGGCT 3′; RPIA-ΔC (nt 568–867 deletion) (forward) 5ʹ ATAATGGCTG AGAGAGTCTACTTTGGGATG 3ʹ and (reverse) 5ʹ AGAAGCATAGCCAGCCACAATCTTCT 3′; RPIA-ΔD (nt 868–936 deletion) (forward) 5ʹ ATAGAATTCACTTCAGCGGAGGCCGGAG 3ʹ and (reverse) 5ʹ AGACTCGAGGTTGATGAATAGGCCTGTGTCC 3ʹ. Transient transfection of the plasmid DNAs was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s manual.Colon cancer tissue staining data were obtained by using the tissue array CDA3 from SUPER BIO CHIPS and the tissue array MC5003a from US Biomax. The slides were incubated with mouse anti-RPIA (1:100) or rabbit anti-β-catenin (1:150) primary antibody at 4°C overnight after the dewax, rehydration, and antigen retrieval steps. The HE staining procedure was performed as outlined in our previous report, and tissues were examined with light microscopy .Total protein was extracted from cells using whole-cell extract (WCE) lysis buffer. Lysates were vibrated for 30 min and centrifuged at 13,200 rpm for 20 min at 4°C. Western blotting was performed as outlined in our previous report , and theRNA was extracted from paired samples of patient tissues, transgenic zebrafish tissue, or cell lines homogenized in TRIzol. cDNA was reverse transcribed from RNA using a High Capacity RNA-to-cDNA Kit . qPCR was performed using an ABI Prism 7500 Sequence Detection System . Gene expression was amplified with the primers listed in Supporting information: Renilla luciferase was used as an internal control. The transfected cells were harvested 48 h post-transfection and lysed by the buffer supplied in the Dual-Glo Luciferase Assay Kit , and luciferase activity in lysates was measured with a luminometer.Cells were transfected with TOPflash (containing a WT TCF binding site) or FOPflash (containing a mutated TCF binding site), which were purchased from Millipore, and Transfected cells grown on cover slips were fixed in 4% paraformaldehyde for 10 min at room temperature and permeabilized in 0.5% Triton for 10 min. After 1 h of blocking in 2% FBS at room temperature, slides were incubated with anti-RPIA or anti-APC primary antibody at 4°C overnight. Secondary antibodies conjugated with Texas Red or FITC were used, and DAPI was used to stain nuclei. The images were scanned and captured with confocal microscopy.RPIA (NM_144563.2) was amplified by PCR with the attB1-F-RPIA and attB2-R-RPIA primer pair using cDNA from the HEK293 cell line as a template. PCR was performed using a KOD FX  and a 994-bp amplicon. The following forward primer was used for Gateway cloning: attB1-F-RPIA (Tm:59°C):5ʹGGGGACAAGTTTGTACAAAAAAGCAGGCTATGCAGCGCCCCGGGCC3ʹ, and the following reverse primer was used for Gateway cloning: attB2-R-RPIA (Tm:58°C):5ʹGGGGACCACTTTGTACAAGAAAGCTGGGTTCAACAGAAAGGCTTCTCCCTCATG3ʹ. PCR comprised the following steps: stage I: 94°C for 5 min; stage II (35 cycles): 95°C for 30 sec, 58°C for 30 sec, and 72°C for 2.5 min; stage III: 72°C for 7 min; and stage IV: 4°C.The coding region of human n = 36) and AB line (WT) zebrafish which served as controls (n = 12) were analyzed in this study.Gateway cloning was performed to generate the final expression construct, namely, pTol2-ifabp: RPIA; myl7: EGFP, using a MultiSite Gateway Three-Fragment Vector Construction Kit (Invitrogen). The transgenic zebrafish model was established via microinjections of the above constructs, which were performed as described elsewhere, and the transgenic fish were selected as described previously . The F2 The zebrafish were maintained at the TZCF under an automated 14:10-h light:dark cycle and a constant temperature of 28°C under continuous flow. MS 222/tricaine methanesulfonate (160 mg/L) were applied for anesthesia.S1 Fig(A) The arbitrary IRS were calculated as compared to the IRS standards shown. Following RPIA IHC in multiple colon cancer tissue arrays  were graded for staining intensity as 1 (weak), 2 (moderate), and 3 (strong) and the percentage of positive cells were scored as 1 (<10%), 2 (11%–50%), 3 (51%–80%), and 4 (>80%). The IRS value is determined by the multiplication of the 2 different scores. Magnification: 200 X. Scale bar: 500 μm. (B) Three siRNA was designed from Invitrogen and pooled for using in colon cancer cell lines. The relative position of siRNA was shown. NCBI reference sequence of RPIA: NM_144563.2. IHC, immunohistochemistry; IRS, immunoreactive score; NCBI, National Center for Biotechnology Information; RPIA, ribose-5-phosphate isomerase A.(TIF)Click here for additional data file.S2 FigCCND1 and CCNE2 in SW480 cells. (H) Overexpression of RPIA increased the mRNA levels of β-catenin target genes CCND1 and CCNE2 in SW480 cells. The statistical significance was calculated by Student t test (** 0.001 < P < 0.01). Data can be found in CCND1, Cyclin D1; CCNE2, Cyclin E2; pcDNA, pcDNA vector control; qPCR, quantitative PCR; RPIA, ribose-5-phosphate isomerase A; si-NC, negative control siRNA; siRNA, small interfering RNA; si-RPIA, RPIA siRNA; TCF, T-cell transcription factor.(A) Knock down of RPIA significantly reduced cell proliferation, and RPIA overexpression enhanced cell proliferation in SW480 cells. Co-treatment of si-RPIA and pcDNA-RPIA rescued the reduction of cellular proliferation which upon knockdown of RPIA in SW480. Cell viability assays were performed by measuring the cells at the second, third, fourth, and fifth days as compared to the first day result of control cells. Control: Co-transfect with scramble RNA and pcDNA empty vector. (B) RPIA knockdown significantly reduced colony formation ability, and RPIA overexpression enhanced colony formation ability in SW480 cells. si-NC: Transfect with scramble siRNA as negative control. Representative images of the colonies were shown on top of the quantification result of colony formation. (C) Knockdown of RPIA reduced β-catenin protein levels as measured by western blotting (left panel) and quantified by image J (middle panel) but did not significantly alter mRNA levels of β-catenin as measured by qPCR (right panel) in SW480 cells. (D) RPIA overexpression increased β-catenin protein levels (left and middle panels) but did not affect β-catenin mRNA levels (right panel) in SW480 cells. (E) Knockdown of RPIA reduced the β-catenin/TCF luciferase reporter activity in SW480 cells. (F) Overexpression of RPIA induced the β-catenin/TCF luciferase reporter activity in SW480 cells. (G) Knockdown of RPIA decreased the mRNA levels of β-catenin target genes (TIF)Click here for additional data file.S3 FigSer9 protein expression levels were up-regulated in cytoplasmic extraction upon overexpression of RPIA-WT, but not upon RPIA-ΔD in SW480 cells. (K) Cell proliferation was measured in RPIA knockdown in SW480 combined with 2.5 mM LiCl or 5 μM CHIR99021, respectively. The statistical significance was calculated by Student t test . Data can be found in (A) Knockdown of RPIA reduced β-catenin protein levels and RPIA overexpression increased β-catenin protein levels in both cytoplasmic and nuclear fractions of SW480 cells. (B) Knockdown of RPIA did not decrease ERK and pERK protein levels which were measured by western blotting in total protein analysis (up panel) in SW480. Conversely, overexpression of RPIA did not increase ERK and pERK protein levels (up panel). In the low panel, both cytoplasmic and nuclear fraction showed that ERK and pERK protein levels were not up-regulated in SW480. (C) Knockdown of RPIA did not decrease EGFR and pEGFR protein levels, which were measured by western blotting in total protein from HCT116 or SW480. (D) To determine the half-life of β-catenin protein, western blots were used to measure the abundance of β-catenin at different time points following the addition of the protein synthesis inhibitor CHX (10 μg/ml) in SW480 cells transfected with either control siRNA or RPIA-siRNA. The lower panels show plots of the relative β-catenin protein level, expressed as a percentage as a function of time after CHX treatment. (E) RPIA-ΔD lost the ability to stabilize β-catenin. Relative β-catenin protein levels as measured by quantification of western blot are shown in SW480 cells. (F) In SW480 cell, the reduced β-catenin levels by RPIA knockdown were rescued by 5 μM of MG132 treatment (left panel). Inhibition of RPIA stimulated ubiquitination of β-catenin (right panel). β-Catenin was precipitated by specific antibody. Coprecipitated ubiquitin levels were examined via western blot with anti-ubiquitin antibody. (G) The phosphorylated β-catenin (at Ser33/Ser37) versus total β-catenin was elevated upon RPIA knockdown. Gel images are shown on the up panel. (H) Overexpression of nondegradable β-catenin can overcome the growth inhibition by RPIA knockdown in SW480 cells. The proliferation fold is compared to pMCV6 transfected control cell at first day. (I) The elevated viability by expression of RPIA was decreased upon ICRT14 (β-catenin inhibitor) treatment. Dose-dependent effects were revealed in SW480. (J) pGSK3β(TIF)Click here for additional data file.S4 Fig(A) Nuclear localization of RPIA was immunostained with an anti-RPIA antibody (green) in SW480 cells with and without overexpression of RPIA. DAPI was used to counterstain nuclei (blue). Scale bar: 50 μm. Co-localization of RPIA with APC or APC with β-catenin in SW480 were shown in fluorescence in the merge result. (B) Left panels: The cell lysates were precipitated by anti-APC, anti-β-catenin and anti-RPIA antibody in SW480 cells. The APC, β-catenin, and RPIA interaction can be increased by RPIA-WT but not by RPIA-ΔD. Right panels: Protein loading input for IP for SW480 cells. The orange boxes indicated those signals were enhanced by RPIA-WT but not in RPIA-ΔD. (C) Model of RPIA-β-catenin-APC interaction in SW480 cell line. APC, adenomatous polyposis coli; RPIA-ΔD, RPIA deletion domain D mutant; RPIA, ribose-5-phosphate isomerase A; RPIA-WT, RPIA wild type.(TIF)Click here for additional data file.S5 Fig(A) The mRNA levels of WT and five deletion mutated-RPIA were analyzed by qPCR. (B) RPIA protein expression pattern was presented by western blot. The definite size is marked with asterisks. (C) The effect on cell proliferation after the expression of RPIA-WT and the different RPIA deleted constructs in SW480 cells. (D) RPIA-ΔD lost the ability to stimulate the TOPflash luciferase construct in SW480 cells. Data can be found in (TIF)Click here for additional data file.S6 Fign = 6) and RPIA Tg fish (n = 18) from three portions of guts. The gene expression levels were analyzed with qPCR. There are extreme data in each group, so they are removed for the statistical analysis. (A) For IB, the number of WT is 5, and the number for RPIA is 17. (B) For MI, the number of WT is 3, and the number for RPIA is 18. (C) For PI, the number of WT is 5, and the number for RPIA is 18. One-year-old RPIA Tg fish (n = 21) and control fish (n = 18) were analyzed for the body weight, body width, intestine length, and body length (D-G). In the elder fish, overexpression of PRIA significantly decreased (D) body weight, (E) body width, and (F) intestine length (G). However, the body length was not affected. The statistical significance was calculated by Student t test . Data can be found in The expression level of β-catenin target genes was analyzed in 5-month-old control fish ((TIF)Click here for additional data file.S1 TableThis table lists the human qPCR primers. qPCR, quantitative PCR.(DOCX)Click here for additional data file.S2 TableThis table lists the zebrafish qPCR primers. qPCR, quantitative PCR; RPIA, ribose-5-phosphate isomerase A.(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S6 Data(XLSX)Click here for additional data file.S7 Data(XLSX)Click here for additional data file.S8 Data(XLSX)Click here for additional data file.S9 Data(XLSX)Click here for additional data file.


The mechanical properties of the hybrid materials and epoxy and carbon fiber (CF) composites were improved significantly as compared to the CF composites made from unmodified epoxy. The reasons could be attributed to the strong interfacial interaction between the CF and the epoxy composites for the existence of carbon nanomaterials. The microstructure and dispersion of carbon nanomaterials were characterized by transmission electron microscopy (TEM) and optical microscopy (OM). The results showed that the dispersion of the hybrid materials in the polymer was superior to other carbon nanomaterials. The high viscosity and shear stress characterized by a rheometer and the high interfacial friction and damping behavior characterized by dynamic mechanical analysis (DMA) indicated that the strong interfacial interaction was greatly improved between fibers and epoxy composites. Remarkably, the tensile tests presented that the CF composites with hybrid materials and epoxy composites have a better reinforcing and toughening effect on CF, which further verified the strong interfacial interaction between epoxy and CF for special structural hybrid materials. FRPet al.  studied  [et al.  suggeste [et al.  reviewed [et al.  introducIn recent years, carbon nanomaterials, especial for MWCNTs  and grapIn our previous study ,20, the Hydroxyl multi-walled carbon nanotubes  were purchased from Shenzhen Nanotech Port Co Ltd. . Hydroxyl graphene nanoplatelets  were purchased from Xiamen Knano Graphene Technology Co. Ltd. . Acryloyl chloride was supplied by J & K Scientific Ltd. . Tetrahydrofuran (THF), 1,4-dioxane and 2,2′-azosiobutyrontrile (AIBN) were purchased from Sinopharm Chemical Reagent Co. Ltd. . Epoxy resin , curing agent , and accelerant  were supplied by the Suzhou Dongwu Glass Instrument Co., LTD. Spread CF tow  was supplied by Toray Industries .The fillers and epoxy and CF composites were prepared according to the national standard GB/T3362-2005. Firstly, we prepared the pure epoxy composite system by adding epoxy resin, curing agent, and catalyst into a breaker . Second2 gas at 50 mL/min was used in the DSC experiment. Rheological measurements were performed at room temperature using a stress-controlled rheometer . The viscosity of the different epoxy composites with different nanoparticle and resin-hardener-accelerator systems were measured with a steady-state flow program, with the shear rate ranging from 0.01 to 100 s−1. The dynamic mechanical properties were tested via dynamic mechanical analyses ). All sample dimensions were 60 mm × 15 mm × 2 mm and were carried out in the torsion rectangular mode with a constant frequency of 1 Hz and a heating rate of 2 °C/min in a temperature range of 30–200 °C. The tensile properties of composites were tested with an Instron3385H tensile tester  with a 2-mm/min tensile speed. The effective length of all samples is 15 mm according to the GB/T1146-2005. The micrography of fracture surface of CF composites was observed via scanning electron microscopy , Beijing, China).The morphologies of carbon nanomaterials were observed via transmission electron microscopy ), with an accelerating voltage of 20 to 30 kV. The dispersion state of different epoxy composites was observed by VH-S30 KEYENCE ultra-depth optical microscope (OM) . The curing reaction of epoxy composites was carried out by the DSC-Q200 differential scanning calorimeter ) with the temperature control procedure: The samples were implemented at a heating speed 10 °C/min from 30–230 °C. Then, the temperature was reduced to 30 °C from 230 °C based on the speed of 30 °C/min and the temperature was then increased to 200 °C at a speed of 10 °C/min. Additionally, NThe dispersion state of various fillers in tetrahydrofuran (THF) is investigated by the images of the suspensions at a filler concentration of 0.1 mg/mL. The dispersion solutions were prepared via 72 h sonication and 1 h sedimentation. Then, the prepared solutions were placed on a microscope slide with a syringe to observe under the optical microscope. gT) curves of epoxy with different fillers are shown in gT of epoxy composites are presented in gT of the epoxy composites was lower than that of the pure epoxy. However, compared to the epoxy composites with different nanoparticles, the curing reaction of the epoxy composites with the hybrid materials started earlier, and the gT was higher than that of the epoxy composites with MWCNTs-OH and GnPs-OH, respectively. The phenomenon can be explained as follows: For the liquid uncured thermosetting polymers, in the curing process, the mobility of the epoxy segments decreased, and the glass gT increased. Generally, when the gT becomes higher than the curing temperature, the reaction terminates due to the “frozen polymer chain” [gT than that of the pure epoxy, due to the enlarged free volume arising from the interface between the fillers and the epoxy resin. The epoxy composites with hybrid materials were observed to have a higher gT than the epoxy composites with MWCNTs-OH or GnPs-OH, respectively. This can be explained by the fact that the interrupted cross-linking networks that form between the resin and the PACl wrapped on the hybrid materials can also lead to an increased gT [The gelation, curing, vitrification can be studied with DSC tests. The curing reaction curves and glass transition temperature  shows the information on the storage modulus (G′), loss modulus (G″), and dissipation factor (tanδ) in the test temperature range. The elastic property and the energy storage of the epoxy composites can be characterized by the storage modulus. The viscous behavior and the energy dissipation of epoxy composites are represented by the loss modulus . As showvs. strain curves. The strength can be represented by the tensile strength, and the toughness often characterized by breaking elongation and integral area of tensile stress The MWCNT and GnP hybrid materials perform a good dispersion in THF according to the TEM images and micrograph, whose 3D structure and polymer bridge inhibit the aggregation of GnPs-OH and MWCNTs-OH. For the nanomaterials/epoxy composites, the DSC results show that fillers could inhibit the mobility of polymer chains and strengthen the interfacial interaction in turn. The rheological behavior of epoxy composites indicates that higher viscosity and higher shear stress also provide evidence for strong interfacial interaction between fillers and epoxy matrices. Similarly, the interfacial friction, damping behavior, and free volume are obtained from DMA to characterize interfacial interaction. For the nanomaterials, epoxy, and CF composites, the CF modified by hybrid materials and EP performs the highest tensile strength and elongation breaking, benefitting from the 3D structural hybrid materials. According to the SEM photos of the fracture surface of CF composites, the hybrid materials and CF and epoxy composites attach with massive EP, owing to the hybrid materials distributed in the epoxy composites homogeneously, and these nanoparticles are proposed to be a “buffer” that can bear and spread external load in the stretching process.


Most cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-β-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs.Two FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) β-catenin expression was analyzed by Western blot analysis; 2) Wnt-β-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of β-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed.We have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced β-catenin/TCF-mediated activity. Additionally, β-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in β-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population.Our results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur during early stages of malignant transformation.The online version of this article (doi:10.1186/s12885-016-2809-9) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality . About 5One of the most critical genes mutated in CRC is the adenomatous polyposis coli (APC) tumor suppressor gene , 2. APC Since APC mutations are detected very early in the adenoma-carcinoma sequence, the APC protein has been suggested to act as a "gatekeeper" of colorectal carcinogenesis, which means that functional loss of APC is a prerequisite for the progression towards malignancy. Around 85 % of all sporadic and hereditary colorectal tumors show loss of APC function . IndividThe APC gene includes a mutation cluster region (MCR) which is prone to mutations. The cell will have a selective advantage for tumor formation when at least one of the mutations (germline or somatic) is located within the MCR region that includes multiple β-catenin binding sites. Indeed, APC mutations in colorectal tumors are distributed non-randomly within the gene , with thMost of our knowledge about the initiation and development of CRC came from studies performed in cancer cells derived from CRC-affected patients . In addiHuman embryonic stem cells (hESCs) have already been proven to be a valuable tool for studying human genetic disorders –25. Pre-The use of spare in-vitro fertilization (IVF)-derived embryos that have been diagnosed by PGD as genetically affected for the generation of hESCs was approved by the Israeli National Ethics Committee (7/04-043). FAP-affected embryos were cultured to the blastocyst stage. At day 6–7 of development, the embryos were micromanipulated to isolate the inner cell mass (ICM) cells for derivation of hESCs as previously described . IsolateOCT4, SSEA-4 and TRA-1-60 by immunofluorescence. FACS analysis of undifferentiated hESCs was performed using Alexa Flour-488 SSEA-3 antibodies (BioLegend) and their respective isotype controls. Samples were analyzed using a BD FACS Canto flow cytometer (BD Biosciences).Two FAP hESC lines were examined in this study: Lis25_FAP1 (FAP1) that we have already described elsewhere  and Lis3Karyotype analysis was performed as previously described . The difFAP1, FAP2 and normal hESC lines were fixed, washed with PBS, permeabilized with PBS containing 0.1 % Triton (PBT) and blocked in 1 % BSA and 0.1 % Triton in PBS for one hour. The cells were then incubated at room temperature with primary antibodies  and further incubated with secondary antibodies . The cell nuclei were stained with 5 μg/ml 4′,6-diamidino-2-phenylindole  or with 5 μM 1,5-bis (2-(di-methylamino)ethylamino)-4,8-dihydroxyanthracene-9,10-dione . The slides were visualized by confocal microscopy or by phase contrast microscopy .Protein was extracted from hESCs grown on matrigel (1:100 in KO-DMEM), using 100 μl lysis bufferX1 (Promega) with a 1 % protease inhibitor cocktail (Sigma). Cell lysates were incubated for 20 min on ice, centrifuged, and the supernatants were separated on 7.5 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to nitrocellulose membranes  using BIO-RAD Mini Trans-Blot Cell. The membranes with the proteins were subjected to blocking solution (0.001 % TWEEN-20 in phosphate buffered solution (PBS) with 5 % low fat milk, Sigma). They were then incubated with primary antibody overnight at 4 °C, and washed with 0.001 % TWEEN-20 in PBS, followed by incubation for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody. After washing, the membranes were exposed to enhanced chemiluminescence detection analysis . The antibodies used were: rabbit anti β-catenin, Santa Cruz Biotechnology; mouse anti-β-actin, Abcam; peroxidase-conjugated goat anti-rabbit and peroxidase-conjugated goat anti-mouse, Jackson Immune Research.Renilla Luciferase Reporter Vectors, to monitor transfection efficiency, mixed with 1.2 μl jetPRIME reagent for 4 h incubation, and then replaced by fresh growth medium. The cells were harvested on ice 48 h later by reporter lysis buffer (Promega) and their luciferase activity was measured by Lumistar Optima (BMG LABTECH) following the manufacturer's instructions. The statistical analysis was performed by Welch's t test. A p value of 0.05 was considered significant.Transfection of undifferentiated hESCs was carried out by a jetPRIME® transfection kit (Polyplus) following the manufacturer's instructions. The cells were seeded on 24-well plates, cover with matrigel  and grown to 60–80 % confluence. Transfection was carried out with 0.6 μg of DNA (pGL3-OT (pTOPFLASH) or pGL3-OF (pFOPFLASH) luciferase reporter constructs containing three copies of either wild-type (WT) or mutated TCF binding element, respectively, and a The partners of couples that underwent IVF treatment for the purpose of PGD of which one of them is a carrier of a pathogenic mutation in the APC gene and had severely affected relatives or aborted fetuses with FAP. At day 3 post-fertilization, embryos at the 6–8 cell stage were biopsied by the aspiration of 1–2 blastomeres from each. The biopsied blastomeres were then subjected to single-cell genetic analysis for the specific APC mutation as well as for 3–6 polymorphic markers flanking the mutation . According to that analysis, embryos diagnosed as carrying the normal allele were transferred back to the uterus to allow implantation and the development of a pregnancy with a healthy fetus. Embryos diagnosed as inheriting the germline APC mutation were donated by the couple for the derivation of hESCs after they signed informed consent for the use of spare PGD embryos for the generation of hESCs, a study approved by the Israeli National Ethics Committee (7/04-043) . A protoDNA was extracted from hESC lines following culture on matrigel (1:100 in KO-DMEM) using the QIAGEN-flexigene DNA kit . DNA extraction was performed according to the manufacturer’s protocol. Amplification of hESCs DNA was performed encompassing the MCR region, the APC gene "hot spots" and the germline mutation. The PCR products were purified by the QIAquick PCR purification kit (QIAGEN) following the manufacturer's instructions, and sequenced using the ABI330XL .The entire APC gene was also sequenced using Pronto Diagnostics kit specifically aimed at sequencing all the coding sequences of the APC gene. The extracted DNA was sent to ProntoLab™, Pronto Diagnostics' molecular services laboratory  which used Multiplicom's  FAP MASTR™ and MID Dx 1–48 for Illumina MiSeq® kits for library preparation. Illumina’s  MiSeq Reagent Kit v2 (500 cycle) was used to run the library on the MiSeq instrument. The FAP MASTR™ kit enables identification of point mutations by complete coverage of all coding sequences of the APC gene. Data analysis was carried out using SeqNext module v4.1.2 of the Sequence Pilot software .The Lis25_FAP1 and Lis30_FAP2 hESC lines were established following PGD for FAP patients by means of approved protocols , 27, 33.The two FAP hESC lines (FAP1 and FAP2) were propagated for a long period of time (>50 passages) in culture without losing their pluripotent properties. Both lines demonstrated a typical morphology of hESCs with a normal karyotype Fig. . AltogetGenetic and epigenetic instability have been strongly associated with various types of cancer. Extended culture of hESCs has already been shown to be associated with genetic instability , 34–36, Undifferentiated FAP-HESCs were propagated for >45 passages and the cell extracts of high passages were compared to those of early passages. In addition, as APC is extremely difficult to detect in minute tissue samples, we assayed its most affected downstream counterpart β-catenin.P < 0.05, Welch's t test)  demonstrated wide fluctuations in β-catenin levels  Fig.  and to est) Fig. . In contst) Fig. .Fig. 4WnInactive β-catenin is usually localized to the membrane or cytoplasm, however, upon activation it translocates and accumulates into the nucleus. We therefore analyzed β-catenin activation also by determining its cellular localization using an anti-β-catenin antibody and confocal laser microscopy. Interestingly, we found that β-catenin was localized to perinuclear structures in most high passage FAP1 hESCs Fig. . In contIn view of the fact that β-catenin in high passage FAP1-hESCs is sequestered to yet unknown cellular structures next to the nucleus, we used RAB11 that was previously shown to co-localize with β-catenin in similar structures . Our resAt this stage we wanted to explore the relationship between the increased β-catenin levels observed following extended culture of FAP1 hESCs and APC mutation. It is well established that the levels of expression and subcellular localization of β-catenin is regulated by the β-catenin destruction complex that includes the APC protein. A mutation in APC will result in a nonfunctional protein product that will lead to increased β-catenin levels. In search of the somatic mutations following extended cultures of FAP1 hESCs, we sequenced the entire MCR region (codons 1250–1450) and the hot spot regions (codons 1450 and 1554) within the APC gene in the FAP1 hESC lines from high passages Fig. . We wereAnother explanation for the differences between FAP1 and FAP2 hESCs may be that mutations in other components of the Wnt cascade, such as in Axin1 or GSK-3β, may have contributed to the high levels of Wnt signaling-mediated transcription in FAP1 hESCs but have not occurred in FAP2 hESC. It has been shown that mice that express an APC protein which lacks the armadillo repeat region, develop significantly more polyps than mice that express this region, very similar to the differences we observed between FAP1 and FAP2 . The molAnother explanation for the increased β-catenin activity in FAP1 hESCs may be the differentiation state of the cells. Wnt/β-catenin signaling was shown to maintain self-renewal under feeder-free conditions in undifferentiated hESCs  while otThe current study describes the establishment of FAP hESCs that carry the germline mutation in the APC gene as a novel human in-vitro model that can be used for studying the first steps in cancer development. To the best of our knowledge, this is the first report on PGD-derived hESC lines that carry mutation in a gene with predisposition for cancer . Human pTaken together, these results demonstrate that our FAP-ESC lines are a unique human in vitro model with a mutation predisposition for cancer that can serve to study the early molecular and cellular events leading from such a single mutation into colorectal cancer.predisposition for cancer. TP53 function is frequently compromised during tumorigenesis as a result of homozygous somatic mutations, which are seen in more than 50 % of human cancers [In the cancer literature several other mutations like TP53 were also shown to cause a  cancers . Single- cancers –51. Amir cancers . This stUsing CRISPR/Cas9 genome engineering we are currently inducing mutations in the second copy of the APC gene as found in human CRC, mimicking normal progression of the disease. Using these innovative approaches with the FAP-hESCs studies here, will enable us to study in vitro, in a human based model, the earliest cellular and molecular events directly caused by the inactivation of APC and how this might lead to cancer initiation in otherwise normal cells. Furthermore, the development of an in vitro model system to study CRC from its earliest stages onward could greatly accelerate efforts toward the development of therapeutics and provide a platform for testing preventative strategies.


Xenopus tropicalis have enabled us to uncover a genome‐wide view of Wnt/β‐catenin signaling in early vertebrate embryos, which challenges previous concepts about molecular mechanisms of Wnt target gene regulation. In this review, we summarize our experimental approaches, introduce the technologies we employed and focus on recent findings about Wnt target gene regulation from Xenopus research. We will also discuss potential functions of widespread β‐catenin binding in the genome that we discovered in this species.Wnt/β‐catenin signaling is an important cell‐to‐cell signaling mechanism that controls gene expression during embryonic development and is critically implicated in human diseases. Developmental, cellular, and transcriptional responses to Wnt signaling are remarkably context‐specific in different biological processes. While nuclear localization of β‐catenin is the key to activation of the Wnt/β‐catenin pathway and target gene expression, the molecular mechanisms of how the same Wnt/β‐catenin signaling pathway induces specific responses remain undetermined. Recent advances in high‐throughput sequencing technologies and the availability of genome information for  Strikin22.1et al., et al., Candidate gene approaches had previously led to the discovery of maternal Wnt‐regulated genes and of the molecular mechanisms controlling their expression  technique in this species .For the purpose of discovering direct zygotic Wnt8a/β‐catenin target genes, we logically defined two criteria for the genes that we wanted to identify: first, that the transcript levels of the genes must be altered (increased or decreased) when the Wnt8a signaling activity is experimentally changed; and second that there must be β‐catenin recruitment to genomic sequences nearby. To this end, we decided to gain a genome‐wide perspective by combining RNA‐seq transcriptome analysis using 2.2wnt8a‐depleted embryos (by wnt8a morpholino (MO) knock down), we also studied gene expression profiles of the embryos, in which the wnt8a knock down was experimentally rescued with injection of a MO‐insensitive wnt8a‐expressing DNA construct ; but importantly, none of the known maternal Wnt target genes, highlighting robust mechanisms that exclude them in order to ensure correct context‐specific responses to Wnt signaling.Our bioinformatics analysis of the resulting RNA‐seq data led to the identification of about 13 clear Wnt8a‐regulated genes Figure a and a f2.3Chromatin Immuno‐Precipitation (ChIP) technique combined with high‐throughput sequencing that enables us to obtain a genome‐wide snapshot of protein–DNA interactions in a particular context . ChIP‐seq analysis turned out to be more challenging than RNA‐seq approaches, as its success highly depends on the availability of a high quality antibody targeting the protein of interest, and on the quantity of chromatin‐bound proteins. In particular, because Xenopus embryos are yolk‐rich, longer fixation times were required and thus additional optimization was necessary for determining appropriate chromatin crosslinking time depending on the stage of development.ChIP‐seq uses the Xenopus tropicalis genome, which were bioinformatically linked to approximately 5,000 nearby genes . A high degree of correlation between those many β‐catenin‐associated sequences with genomic locations previously identified as regulatory sequences (e.g. promoters and enhancers) confirmed this β‐catenin chromatin‐association to be due to specific binding.β‐catenin ChIP‐seq Figure b is a po2.4hoxd1, Figure Our combined analysis revealed that the number of β‐catenin‐associated gene loci is considerably larger than that of Wnt‐regulated genes in post‐MBT embryos Figure c. Those Our conclusions further became firm after subsequent analysis comparing β‐catenin recruitment to context‐specific genes for the different contexts before and after the MBT. We experimentally increased or decreased the Wnt8a signaling activity in post‐MBT embryos and studied transcription levels of the “wrong” genes, i.e., of maternal Wnt‐regulated genes that were uncovered to have β‐catenin recruitment by our β‐catenin ChIP‐seq analysis in post‐MBT embryos. In these post‐MBT experimental conditions, as expected, the transcription of these maternal Wnt target genes remained unaffected. However, the β‐catenin binding level was significantly increased by the enhanced Wnt8a activity, demonstrating that Wnt‐regulated β‐catenin recruitment does not necessarily lead to transcriptional activation of associated genes. This finding further prompted us to test whether maternal β‐catenin also has any potential to control direct zygotic Wnt8a target genes before the MBT. Interestingly, we observed that maternal β‐catenin was precociously associated with zygotic Wnt8a target gene loci in pre‐MBT embryos; that this β‐catenin association was regulated by maternal Wnt signaling even though as expected there was no transcriptional regulation of these zygotic Wnt8a target genes. Taken together, these findings clearly show that Wnt‐regulated β‐catenin recruitment is not sufficient for transcriptional activation in the “wrong” developmental context (see Figure 2.5et al., What is so special about the transcriptionally regulated Wnt8a/β‐catenin target genes? Since direct maternal Wnt/β‐catenin target genes are regulated by combinatorial Wnt and Activin/Smad2 signaling (Crease, Dyson, & Gurdon, We however uncovered that rather than sharing a single ventral mesoderm‐specific mechanism, Wnt8a target genes turned out to have more gene‐specific regulatory mechanisms. For some of the Wnt8a target genes, BMP signaling is indispensable, and for some others FGF signaling is essential. Analysis of expression domains showed that BMP‐ or FGF‐dependent Wnt8a target genes are expressed in different tissues of early gastrulae. Overexpression and inhibition experiments demonstrated that these signaling pathways provide spatially different, partially overlapping contexts for Wn8a target genes to respond to Wnt8a/β‐catenin signaling.Although the BMP or FGF signaling pathways are required for Wnt8a target gene expression, neither BMP nor FGF signaling controls β‐catenin activation or accessibility to chromatin. The context‐specific Wnt target gene regulation by these pathways occurs separately from β‐catenin recruitment (see Figure Xenopus development. Alternatively, these co‐regulating BMP and FGF pathways may more independently influence transcriptional activation of Wnt/β‐catenin target genes by promoting the removal of any negative regulators or the addition of positive regulators to separate cis‐regulatory sequences of the same gene.The detailed molecular mechanisms of this context‐specific Wnt target gene regulation remain unclear. So far, a number of β‐catenin‐interacting proteins have been reported (Mosimann, Hausmann, & Basler, 33.1et al., neurod1 (Kuwabara, Hsieh, Muotri, Yeo, Warashina, Lie, … Gage, et al., The first proposal involves binding of a transcription factor through specific DNA motifs but this is not immediately associated with direct transcription at nearby sites. Only at later stages or in other tissues does this binding lead to expression of those genes when it is integrated with binding by additional transcription factors. Interestingly, our β‐catenin‐binding sites included many genes activated at later stages by Wnt signaling [e.g., Another proposal involves a buffering system through binding to many relatively accessible sites in the chromatin (cf. Lin and Riggs, A further proposal involves chromatin reorganization. Pioneer transcription factors can also bind to otherwise inaccessible sites of closed chromatin (Iwafuchi‐Doi & Zaret, As noted above, our β‐catenin ChIP‐seq has revealed β‐catenin recruitment to a large number of genomic regions, which vastly exceed the number of Wnt8a‐regulated genes in this context. Genome‐wide binding outside of direct transcriptional regulation has been observed for other transcription factors and in different systems (MacQuarrie, Fong, Morse, & Tapscott, et al., Xenopus will again be ideal for such studies.Genome‐wide β‐catenin recruitment could be involved in some of these proposed molecular mechanisms. However, it is noted that β‐catenin is not a DNA‐binding transcription factor. Differences in the contribution of β‐catenin binding could change depending on the DNA‐binding transcription factors that β‐catenin interacts with. Generally, TCF/LEF is the main partner of β‐catenin for recruitment to chromatin (reviewed by Hoppler & Waterman, 4Conventional research on Wnt signaling has logically focused on identifying mechanisms that are universally used in different contexts. However, our understanding is still limited regarding why certain Wnt target genes remain transcriptionally refractory to Wnt signaling activation in one context but can be activated by Wnt signaling in another context.Xenopus genome has challenged this concept and brought about a new paradigm for a Wnt‐regulated transcriptional mechanism. This new paradigm is not only relevant to embryogenesis but also to regeneration and cancer, as well as other Wnt‐related diseases, where a change in cellular contexts may induce expression of genes with precocious β‐catenin binding. The observed widespread β‐catenin binding may also contribute to yet undetermined functions, such as chromatin reorganization and signaling buffering mechanisms. Further investigation of context‐specific Wnt target gene regulation and the role of widespread β‐catenin recruitment in the genome will further benefit from the use of new technologies, such as genome editing, combined with now well‐established high‐throughput sequencing approaches in the experimentally accessible Xenopus model system.Nuclear β‐catenin recruitment has been considered an instant link to transcriptional activation of Wnt target genes. However, the finding of widespread β‐catenin recruitment in the


Wnt/β-catenin signaling is extremely important for proper kidney development. This pathway is also upregulated in injured renal tubular epithelia, both in acute kidney injury and chronic kidney disease. The renal tubular epithelium is an important target of kidney injury, and its response (repair versus persistent injury) is critical for determining whether tubulointerstitial fibrosis, the hallmark of chronic kidney disease, develops. This review discusses how Wnt/β-catenin signaling in the injured tubular epithelia promotes either repair or fibrosis after kidney injury. There is data suggesting that epithelial Wnt/β-catenin signaling is beneficial in acute kidney injury and important in tubular progenitors responsible for epithelial repair. The role of Wnt/β-catenin signaling in chronically injured epithelia is less clear. There is convincing data that Wnt/β-catenin signaling in interstitial fibroblasts and pericytes contributes to the extracellular matrix accumulation that defines fibrosis. However, some recent studies question whether Wnt/β-catenin signaling in chronically injured epithelia actually promotes fibrosis or repair. The human kidney is a highly vascular organ that removes metabolic wastes and excess fluid, maintains acid/base balance, regulates electrolytes, and produces hormones such as renin and erythropoietin that modulate blood pressure and red blood cell production, respectively. Each human kidney contains 500,000–2,000,000 nephrons, the basic functional unit . The nepWnt/β-catenin signaling in renal injury has attracted much attention both for its potential to promote tubular repair but also as a potential target for fibrosis. There are 19 mammalian Wnt ligands that bind to frizzled and LRP5,6 co-receptors to inhibit the β-catenin destruction complex composed of axin, adenomatous polyposis coli (APC), glycogen synthase kinase-3β (GSK-3β), and casein kinase . Wnt/β-cSeveral of the Wnt ligands are expressed during renal development, and reporter mice for β-catenin activity show increased activity during nephron development ,10,11. IMany of the growth factors and gene expression patterns involved in renal development  are recapitulated after injury as the kidney rebuilds . Thus, aWnt ligands play an important role in this epithelial/mesenchymal communication. Expression of Wnt4, Wnt2b, Wnt7b, Wnt6, Wnt9b, and Wnt11 have been detected in the developing kidney, and a LEF/TCF reporter  shows activity in the distal UB tips as well as the adjacent condensing MM . ConvincGenetic studies in mice reveal that both β-catenin deletion and over-expression  lead to aberrant renal development. The mechanisms whereby these developmental abnormalities occur provide insights into the effects of Wnt/β-catenin signaling on renal tubular epithelia. Deleting β-catenin specifically in the UB using homeobox B7(Hoxb7)-Cre led to a range of phenotypes from renal agenesis to cystic/dysplastic kidneys with the severity dependent upon the degree of recombination ,23. In tSIX Homeobox 2(Six2)-Cre was used to selectively delete β-catenin in the MM that led to a smaller nephrogenic zone and fewer nephrons . The remWnt/β-catenin signaling is clearly important for renal development, but the amount of signaling as well as spacial and temporal expression are critical. To achieve tight control over Wnt/β-catenin signaling, several modulators of this pathway likely exist. One inhibitor of Wnt in early MM is the transcription factor Six2. The uninduced MM has multipotent progenitor cells, and Six2 was shown to maintain this population in a self-renewing progenitor state, whereas Wnt signaling committed these cells to becoming nephron progenitors . An endoAs mentioned above, Wnt/β-catenin signaling is minimal in the uninjured adult kidney but becomes upregulated in injured tubules during acute and chronic renal injury. Wnt4 and Wnt9b play prominent roles in renal development, but their roles in injured tubular cells may be less important. One study showed increased proximal tubular expression of Wnt4 after ischemia reperfusion (I/R) , a classAcute kidney injury primarily targets the renal epithelium, particularly the proximal tubules, and several pre-clinical studies indicate that epithelial Wnt/β-catenin signaling plays a protective role early in injury though its role at later time points requires further investigation. Wnt/β-catenin signaling can have potent effects on the epithelial cell cycle which is crucial to repair after AKI. Proximal tubules with Wnt/β-catenin activity also stained for proliferating cell nuclear antigen (PCNA) 24 h after I/R, indicating that these were actively dividing cells . In LLC-Wnt/β-catenin also mitigates AKI by reducing apoptosis and increasing survival in injured renal tubules. Tubule-specific ablation of β-catenin led to increased injury in mice subjected to two models of AKI: I/R and folic acid nephropathy . These c2-induced AKI [Several groups have shown that suppressing GSK-3β, the protein that targets β-catenin for proteosomal degradation, also mitigates AKI. Genetic inhibition of GSK-3β in murine proximal tubules reduced apoptosis and mortality following HgCluced AKI . Furtheruced AKI . TDZD-8 uced AKI . Anotheruced AKI . This deuced AKI . It is tDuring AKI, tubular epithelia are susceptible to apoptosis/necrosis, and there is much interest in understanding how tubular repair and replacement of these lost cells occurs. Many groups are successfully differentiating human embryonic stem cells to form renal tubules and other structures, which has exciting promise in the area of tissue regeneration ,42. HoweWnt/β-catenin signaling likely plays an important role in these proliferating tubular epithelial cells. Wnt-responding cells were lineage traced by using a tamoxifen-inducible Cre under the axis inhibition protein 2 (Axin2) promoter crossed with the mT/mG “tomato” reporter mouse to mark Wnt-responsive  cells with GFP . After AWnt/β-catenin signaling is increased in injured epithelia from both murine CKD models and human biopsies. Unlike AKI, the role of this signaling in repair versus fibrosis progression is less clear. Many studies examining Wnt/β-catenin in rodent models of CKD have done so using systemic inhibitors which have shown improvement in tubulointerstitial fibrosis ,50,51,52Another complicating factor regarding systemic Wnt/β-catenin inhibitors is that certain widely used inhibitors such as ICG-001 actually modulate rather than block signaling. ICG-001 selectively inhibits β-catenin’s interactions with CBP, and recent work with Tregs also demonstrates reduced β-catenin binding to TCF ,55,56. CGiven the divergent effects of Wnt/β-catenin on epithelial cells versus fibroblasts, studies examining this signaling pathway specifically in injured renal epithelia are particularly informative. Tubule-specific deletion of β-catenin (using kinesin spindle protein (Ksp)-Cre) did not have much effect on renal fibrosis after the UUO model of CKD . These cSeveral β-catenin-mediated epithelial effects that have been demonstrated in vitro might promote tubulointerstitial fibrosis B. Wnt/β-There is some additional evidence that epithelial Wnt/β-catenin signaling may be protective in CKD. Wnt6 expression was dysregulated in the tubulointerstitium of patients with diabetic nephropathy and tubules of mice after UUO . In vitrIn summary, the Wnt/β-catenin signaling pathway is critical for proper nephron development and is re-activated in the injured renal tubular epithelium and, specifically, the proliferating tubular progenitors that repopulate the tubule after injury. Several studies suggest that epithelial Wnt/β-catenin is protective in the context of AKI, potentially through anti-apoptotic or pro-proliferative effects. Whether epithelial Wnt/β-catenin is friend or foe in CKD requires further study, and the answer likely depends upon temporal factors and/or the amount of signaling. There may be an important dose-dependent effect whereby a modest increase in epithelial Wnt/β-catenin signaling is beneficial, but augmenting this pathway too much promotes a fibrotic response. It is also important to understand that various inhibitors  modulate Wnt/β-catenin signaling rather than cleanly augment or suppress this pathway. To clarify the role of Wnt/β-catenin signaling in chronically injured epithelia, future studies should alter this signaling pathway in a cell-specific way after the acute phase of injury so that its role in acute versus chronic injury can be discerned. Additionally, if Wnt/β-catenin signaling is enhanced, it should be done so in a way that just affects epithelial signaling , and graded responses should be measured to consider potential dose-dependent responses.Other signaling pathways such as TGF-β and the renin-angiotensin system, also upregulated in chronic injury, have been shown to augment Wnt/β-catenin signaling ,70,71,72