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Adenomatous polyposis coli (APC) truncations occur in many colorectal cancers and are often associated with immune infiltration. The aim of this study was to determine whether a combination of Wnt inhibition with anti-inflammatory (sulindac) and/or proapototic (ABT263) drugs can reduce colon adenomas. Apcmin/+ and doublecortin-like kinase 1 (Dclk1)Cre/+;Apcfl/fl mice were exposed to dextran sulphate sodium (DSS) in their drinking water to promote the formation of colon adenomas. Mice were then treated with either a Wnt-signaling antagonist pyrvinium pamoate (PP), an anti-inflammatory agent sulindac or proapoptotic compound ABT263 or a combination of PP+ABT263, or PP+sulindac. Colon adenoma frequency, size, and T-cell abundance were measured. DSS treatment resulted in significant increases in colon adenoma number (P < 0.001, n > 5) and burden in Apcmin/+ (P < 0.01, n > 5) and Dclk1Cre/+;Apcfl/fl (P < 0.02, n > 5) mice. There was no effect on adenomas following treatment with PP in combination with ABT263. Adenoma number and burden were reduced with PP+sulindac treatment in Dclk1Cre/+;Apcfl/fl mice (P < 0.01, n > 17) and in Apcmin/+ mice (P < 0.001, n > 7) treated with sulindac or PP+sulindac with no detectable toxicity. PP treatment of Apcmin/+ mice increased the frequency of CD3+ cells in the adenomas. The combination of Wnt pathway inhibition with sulindac was more effective in Dclk1Cre/+;Apcfl/fl mice and provides an opportunity for killing Apc-mutant colon adenoma cells, indicating a strategy for both colorectal cancer prevention and potential new treatments for patients with advanced colorectal cancer. Outcomes from the results of this study may be translatable to the clinic for management of FAP and other patients with a high risk of developing colorectal cancer.Significance:Colorectal cancer is one of the most common cancers worldwide with limited therapeutic options. APC and other Wnt signaling mutations occur in the majority of colorectal cancers but there are currently no Wnt inhibitors in the clinic. The combination of Wnt pathway inhibition with sulindac provides an opportunity for killing Apc-mutant colon adenoma cells and suggests a strategy for colorectal cancer prevention and new treatments for patients with advanced colorectal cancer.

Adenoma tumour development in Apcmin/+ and Dclk1Cre/+;Apcfl/fl mice

Adenomatous polyposis coli (APC) truncations occur in many colorectal cancers and are often associated with immune infiltration. The aim of this study was to determine whether a combination of Wnt inhibition with anti-inflammatory (sulindac) and/or proapototic (ABT263) drugs can reduce colon adenomas. Apcmin/+ and doublecortin-like kinase 1 (Dclk1)Cre/+;Apcfl/fl mice were exposed to dextran sulphate sodium (DSS) in their drinking water to promote the formation of colon adenomas. Mice were then treated with either a Wnt-signaling antagonist pyrvinium pamoate (PP), an anti-inflammatory agent sulindac or proapoptotic compound ABT263 or a combination of PP+ABT263, or PP+sulindac. Colon adenoma frequency, size, and T-cell abundance were measured. DSS treatment resulted in significant increases in colon adenoma number (P < 0.001, n > 5) and burden in Apcmin/+ (P < 0.01, n > 5) and Dclk1Cre/+;Apcfl/fl (P < 0.02, n > 5) mice. There was no effect on adenomas following treatment with PP in combination with ABT263. Adenoma number and burden were reduced with PP+sulindac treatment in Dclk1Cre/+;Apcfl/fl mice (P < 0.01, n > 17) and in Apcmin/+ mice (P < 0.001, n > 7) treated with sulindac or PP+sulindac with no detectable toxicity. PP treatment of Apcmin/+ mice increased the frequency of CD3+ cells in the adenomas. The combination of Wnt pathway inhibition with sulindac was more effective in Dclk1Cre/+;Apcfl/fl mice and provides an opportunity for killing Apc-mutant colon adenoma cells, indicating a strategy for both colorectal cancer prevention and potential new treatments for patients with advanced colorectal cancer. Outcomes from the results of this study may be translatable to the clinic for management of FAP and other patients with a high risk of developing colorectal cancer. Significance: Colorectal cancer is one of the most common cancers worldwide with limited therapeutic options. APC and other Wnt signaling mutations occur in the majority of colorectal cancers but there are currently no Wnt inhibitors in the clinic. The combination of Wnt pathway inhibition with sulindac provides an opportunity for killing Apc-mutant colon adenoma cells and suggests a strategy for colorectal cancer prevention and new treatments for patients with advanced colorectal cancer.

Supplementary Methods and Materials from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Supplementary Figures 1-3 from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Supplementary Tables 1-2 from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

The adenomatous polyposis coli (APC) tumor suppressor protein is associated with the regulation of Wnt signaling; however, APC also controls other cellular processes including the regulation of cell adhesion and migration. The expression of full-length APC in SW480 colorectal cancer cells (SW480+APC) not only reduces Wnt signaling, but increases membrane E-cadherin and restores cell-cell adhesion. This report describes the effects of full-length, wild-type APC (fl-APC) on cell-cell adhesion genes and p120-catenin isoform switching in SW480 colon cancer cells: fl-APC increased the expression of genes implicated in cell-cell adhesion, whereas the expression of negative regulators of E-cadherin was decreased. Analysis of cell-cell adhesion-related proteins in SW480+APC cells revealed an increase in p120-catenin isoform 3A; similarly, depletion of APC altered the p120-catenin protein isoform profile. Expression of ESRP1 (epithelial splice regulatory protein 1) is increased in SW480+APC cells, and its depletion results in reversion to the p120-catenin isoform 1A phenotype and reduced cell-cell adhesion. The ESRP1 transcript is reduced in primary colorectal cancer, and its expression correlates with the level of APC. Pyrvinium pamoate, which inhibits Wnt signaling, promotes ESRP1 expression. We conclude that re-expression of APC restores the cell-cell adhesion gene and posttranscriptional regulatory programs leading to p120-catenin isoform switching and associated changes in cell-cell adhesion.

Forebrain development in vertebrates is regulated by transcription factors encoded by homeobox, bHLH and forkhead gene families throughout the progressive and overlapping stages of neural induction and patterning, regional specification and generation of neurons and glia from central nervous system (CNS) progenitor cells. Moreover, cell fate decisions, differentiation and migration of these committed CNS progenitors are controlled by the gene regulatory networks that are regulated by various homeodomain-containing transcription factors, including but not limited to those of thePax(paired),Nkx,Otx(orthodenticle),Gsx/Gsh(genetic screened), andDlx(distal-less) homeobox gene families. This comprehensive review outlines the integral role of key homeobox transcription factors and their target genes on forebrain development, focused primarily on the telencephalon. Furthermore, links of these transcription factors to human diseases, such as neurodevelopmental disorders and brain tumors are provided.

Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications. We have developed a low-viscosity matrix (LVM) suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine. Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices, with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers. We demonstrate the utility of LVM suspension culture for organoid and tumoroid bioreactor applications and biobanking, as well as tumoroid high-throughput drug sensitivity testing. These methods provide opportunities for the study and use of patient-derived organoids and tumoroids from the large intestine., Given the practical limitations of solid matrix-based protocols in organoid culture, Yumiko Hirokawa et al. assess the ability of low-concentration Matrigel conditions to promote intestinal organoid growth. Their results suggest that a low-viscosity culture system can improve live cell yield compared to the existing dome method, while maintaining similar morphology, and represents a useful approach for high-throughput applications of organoids.

The majority of colon adenomas harbor genetic mutations in the APC gene. APC mutation leads to changes in Wnt signalling and cell-cell adhesion: as a consequence, intestinal crypt budding increases and the excess crypts accumulate to form adenomas that progress to colon cancer. When cultured with Wnt, R-spondin, EGF, Noggin, myofibroblast conditioned medium and Matrigel, crypts from normal mouse colon mucosa form crypt-producing organoids and can be passaged continuously. Under the same culture and passage conditions, crypts isolated from colon adenomas derived from Apcmin/+ mice typically grow as spheroidal cysts and do not produce crypts. The adenoma organoid growth requires EGF, but not Wnt, R-spondin or Noggin. However, when mouse colon adenoma spheroids are grown for more than 10 days in the presence of EGF, crypt formation occurs. EGF, EREG, β-cellulin, Neuregulin-1 or AREG are sufficient for initiating crypt formation, however, neuregulin-1 is more potent than the other EGF-family members. EGFR and ErbB2 inhibitors both prevent crypt formation in adenoma cultures. Either EGFR:ErbB2 or ErbB3:ErbB2 signalling is sufficient to initiate adenoma crypt budding and elongation. ErbB2 inhibitors may provide a therapeutic avenue for controlling and ablating colon adenomas.

Colorectal cancer (CRC) is driven by a small set of oncogenic and tumor suppressor mutations. However, different combinations of mutations often lead to poor tumor responses to individual anti-cancer drugs. We have investigated the anti-proliferative and in vitro cytotoxic activity of pair-wise combinations of inhibitors which target specific signalling pathways. Colon cancer cells in non-adherent cultures were killed more effectively by combinations of pyrvinium pamoate (a Wnt pathway inhibitor) and ABT263 (a pro-apoptotic Bcl-2 family inhibitor) or Ly29004 (a PI3kinase inhibitor). However, in a mouse xenograft model, the formulation and toxicity of the ABT737/PP combination prevent the use of these drugs for treatment of tumors. Fortunately, oral analogues of PP (pyrvinium phosphate, PPh) and ABT737(ABT263) have equivalent activity and can be used for treatment of mice carrying SW620 colorectal cancer xenografts. The PPh/ABT263 induced SW620 tumor cell apoptosis and reduced the rate of SW620 tumor growth. Combinations of Wnt signaling inhibitors and specific inhibitor of pro-survival proteins should be considered for the treatment of precancerous colon adenomas and advanced colorectal cancers with APC mutations.

Truncating mutations in the tumour suppressor gene APC occur frequently in colorectal cancers and result in the deregulation of Wnt signalling as well as changes in cell-cell adhesion. Using quantitative imaging based on the detection of membrane-associated E-cadherin, we undertook a protein coding genome-wide siRNA screen to identify genes that regulate cell surface E-cadherin in the APC-defective colorectal cancer cell line SW480. We identified a diverse set of regulators of E-cadherin that offer new insights into the regulation of cell-cell adhesion, junction formation and genes that regulate proliferation or survival of SW480 cells. Among the genes whose depletion promotes membrane-associated E-cadherin, we identified ZEB1, the microRNA200 family, and proteins such as a ubiquitin ligase UBE2E3, CDK8, sorting nexin 27 (SNX27) and the matrix metalloproteinases, MMP14 and MMP19. The screen also identified 167 proteins required for maintaining E-cadherin at cell-cell adherens junctions, including known junctional proteins, CTNND1 and CTNNA1, as well as signalling enzymes, DUSP4 and MARK2, and transcription factors, TEAD3, RUNX2 and TRAM2. A better understanding of the post-translational regulation of E-cadherin provides new opportunities for restoring cell-cell adhesion in APC-defective cells.

Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67–negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells. Cancer Res; 71(10); 3709–19. ©2011 AACR.

Asymmetric stem cell divisions controlled by Apc in the intestinal crypt result in regulated, anisotropic movement of daughter cells away from the niche., The stem cells (SCs) at the bottom of intestinal crypts tightly contact niche-supporting cells and fuel the extraordinary tissue renewal of intestinal epithelia. Their fate is regulated stochastically by populational asymmetry, yet whether asymmetrical fate as a mode of SC division is relevant and whether the SC niche contains committed progenitors of the specialized cell types are under debate. We demonstrate spindle alignments and planar cell polarities, which form a novel functional unit that, in SCs, can yield daughter cell anisotropic movement away from niche-supporting cells. We propose that this contributes to SC homeostasis. Importantly, we demonstrate that some SC divisions are asymmetric with respect to cell fate and provide data suggesting that, in some SCs, mNumb displays asymmetric segregation. Some of these processes were altered in apparently normal crypts and microadenomas of mice carrying germline Apc mutations, shedding new light on the first stages of progression toward colorectal cancer.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are common in both inherited and sporadic forms of colorectal cancer (CRC), and are associated with dysregulated Wnt signaling. Colon carcinoma SW480 cells restored with stable expression of wild-type APC (SW480APC cells) exhibit attenuated Wnt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW480 and SW480APC cells to examine the effects of restored APC on exosome protein expression. A salient finding of our study was the unique expression of the Wnt antagonist Dickkopf-related protein 4 (DKK4) in SW480APC, but not parental SW480 cell-derived exosomes. Upregulation of DKK4 in SW480APC cells was confirmed by semiquantitative RT-PCR, immunoblotting, and immunogold electron microscopy. Analysis of the DKK4 gene promoter by methylation-specific PCR revealed reduced methylation in SW480APC cells, while RT-PCR demonstrated the downregulation of DNMT-3a, compared to the parental cell line. Our discovery of exosome-mediated secretion of DKK4 opens up the possibility that exosomal DKK4 may be a mechanism used by epithelial colon cells to regulate Wnt signaling which is lost during CRC progression.

β-catenin is a member of the armadillo repeat family of proteins and has important functions in cell-cell adhesion and Wnt signalling. Different protein species of β-catenin have been shown to exist in the cell and the relative proportions of these species are altered upon stimulation of cells with Wnt-3a (Gottardi and Gumbiner, 2004). In order to determine whether posttranslational modifications (PTMs) of β-catenin underlie these different protein species, we have used 2DE separation and immunoblotting with an antibody specific for β-catenin. High-resolution separation of differentially modified species of β-catenin in 2DE required the addition of ASB-16, a zwitterionic detergent that can solubilise integral membrane proteins. ASB-16 was also necessary for focussing of other armadillo repeat proteins, such as γ-catenin and p120-catenin. 2DE using ASB-16 allowed detection of a previously unreported phosphorylation site in the transcriptionally active form of β-catenin that binds to GST-Tcf in response to Wnt signalling.

The adenomatous polyposis coli (APC) tumour suppressor is a multifunctional protein involved in the regulation of Wnt signalling and cytoskeletal dynamics. Little is known about how APC controls these disparate functions. In this study, we have used APC- and axin-fluorescent fusion proteins to examine the interactions between these proteins and show that the functionally distinct populations of APC are also spatially separate. Axin-RFP forms cytoplasmic punctate structures, similar to endogenous axin puncta. Axin-RFP recruits beta-catenin destruction complex proteins, including APC, beta-catenin, glycogen synthase kinase-3-beta (GSK3-beta) and casein kinase-1-alpha (CK1-alpha). Recruitment into axin-RFP puncta sequesters APC from clusters at cell extensions and this prevents its microtubule-associated functions. The interaction between APC-GFP and axin-RFP within the cytoplasmic puncta is direct and dramatically alters the dynamic properties of APC-GFP. However, recruitment of APC to axin puncta is not absolutely required for beta-catenin degradation. Instead, formation of axin puncta, mediated by the DIX domain, is required for beta-catenin degradation. An axinDeltaDIX mutant did not form puncta, but still mediated recruitment of destruction complex proteins and phosphorylation of beta-catenin. We conclude that there are distinct pools of APC and that the formation of axin puncta, rather than the axin/APC complex, is essential for beta-catenin destruction.

Recombinant proteins, commonly expressed in fusion with an affinity tag to facilitate purification, are often used as immunogens for polyclonal antibody production. Careful immunopurification of the antibody product is often the key to obtaining a high-specificity polyclonal antibody against the protein domain of interest. This study describes the purification and characterization of such an antibody directed against the adenomatous polyposis coli (APC) tumour suppressor. We used a combination of affinity chromatography and biosensor analysis to optimize and monitor antibody purification. This antibody was then characterized by immunoprecipitation, proteomic analyses and immunofluorescence staining and shown to be a valuable reagent for the study of APC biology. Using this antibody we successfully isolated and identified APC, using MS/MS, from transfected cell lines. A novel phosphorylation site on APC was identified at ser 1436. Similar strategies involving multiple immuno-affinity steps coupled with surface plasmon resonance (SPR), immunoprecipitation proteomic and immunofluorescence analyses should be generally applicable for the purification and characterization of other polyclonal antibodies.

A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST−β-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST- immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC−MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads. Keywords: biosensor micro-affinity purification • proteomics • APC • E-cadherin • β-catenin

Most colon cancers arise from somatic mutations in the tumor suppressor gene APC (adenomatous polyposis coli), and these mutations cause constitutive activation of the Wnt–to–β-catenin pathway in the intestinal epithelium. Because Wnt–β-catenin signaling is required for homeostasis and regeneration of the adult intestinal epithelium, therapeutic targeting of this pathway is challenging. We found that genetic activation of the cytokine-stimulated pathway mediated by the receptor gp130, the associated Jak (Janus kinase) kinases, and the transcription factor Stat3 (signal transducer and activator of transcription 3) was required for intestinal regeneration in response to irradiation-induced damage in wild-type mice and for tumorigenesis in Apc-mutant mice. Systemic pharmacological or partial genetic inhibition of gp130-Jak-Stat3 signaling suppressed intestinal regeneration, the growth of tumors in Apc-mutant mice, and the growth of colon cancer xenografts. The growth of Apc-mutant tumors depended on gp130-Jak-Stat3 signaling for induction of the polycomb repressor Bmi-1, and the associated repression of genes encoding the cell cycle inhibitors p16 and p21. However, suppression of gp130-Jak-Stat3 signaling did not affect Wnt–β-catenin signaling or homeostasis in the intestine. Thus, these data not only suggest a molecular mechanism for how the gp130-Jak-Stat3 pathway can promote cancer but also provide a rationale for therapeutic inhibition of Jak in colon cancer.

The coiled coil is a common structural motif found both as the dominant structure in fibrous proteins and as an oligomerization domain in a variety of cytoskeletal and extracellular matrix proteins. Coiled-coils typically consist of two to four helices that are supercoiled around one another in either parallel or antiparallel orientations. In the past few years our knowledge of the structure and specificity of coiled coil interactions has increased, allowing the de novo design and preparation of coiled-coils with well-defined structure and specificity. Indeed, the design and synthesis of a peptide that binds specifically to a single coiled-coil-containing protein, adenomatous polyposis coli (APC) has been reported. We have optimized solid-phase synthesis techniques to produce a modified form of the anti-APC peptide that contains a biotin moiety specifically placed so as to allow selective orientation onto the surface of a biosensor or affinity support. These peptide surfaces have been used to both monitor and purify APC and APC complexes from cellular extracts.

Large numbers of colon tumors stem from mutations in the gene coding for the production of the adenomatous polyposis coli (APC) tumor suppressor protein. This protein contains a coiled-coil N-terminal domain that is known to be responsible for homodimerization. Previous work by others has led to the design of a specific 54-residue anti-APC peptide (anti-APCp1) that dimerizes preferentially with this domain. We have undertaken the chemical synthesis of a modified form of this peptide (anti-APCp2) that bears a biotin moiety at its N-terminus for use in subsequent ligand-binding analysis studies. The peptide was subjected to comprehensive chemical characterization to confirm its purity. Secondary structural analysis by circular dichroism spectroscopy and Fourier transform infrared spectroscopy indicated that the peptide could assume a wide range of potential conformations, depending upon the precise microenvironment. Significantly, a stable alpha-helical structure was generated when the solvent conditions supported intramolecular salt-bridge formation along the helix barrel. The biotinylated anti-APCp2 was immobilized onto a streptavidin sensor surface, in a specific orientation leaving all amino acids available to form a coiled structure. In one experiment, injection of colonic cell lysate extracts (LIM1215) onto a size-exclusion column resulted in the isolation of a high molecular mass protein peak (> 600 kDa) that reacted specifically with the immobilized anti-APCp2 on the biosensor surface. In another experiment, a high molecular mass protein (M(r) > 250 kDa on SDS-PAGE) could be specifically immunoprecipitated from this peak using either the anti-APCp2 peptide or an anti-APC polyclonal antibody. This demonstrates the specific interaction between the anti-APCp2 peptide and native APC and highlights the potential use of the former peptide in a multidimensional micropreparative chromatographic/biosensor/proteomic protocol for the purification of APC alone and APC complexed with different biopolymers in various cell lines, and stages of tumor development.

The A-kinase-anchoring protein AKAP79 co-ordinates the location of cAMP-dependent protein kinase, phosphatase 2B (PP2B/calcineurin) and protein kinase C (PKC) at postsynaptic sites in neurons. In this report we focus on the mechanism of interaction between AKAP79 and PKC. We show that neither lipid activators nor kinase activation are required for association with AKAP79. The anchoring protein binds and inhibits the conserved catalytic core of PKCbetaII. AKAP79 also associates with conventional, novel and atypical isoforms of PKC in vitro and in vivo, and immunofluorescence staining of rat hippocampal neurons demonstrates that the murine anchoring-protein homologue AKAP150 is co-distributed with PKCalpha/beta, PKCepsilon or PKCiota. Binding of the AKAP79(31-52) peptide, which inhibits kinase activity, exposes the pseudosubstrate domain of PKCbetaII, allowing endoproteinase Arg-C proteolysis in the absence of kinase activators. Reciprocal experiments have identified two arginine residues at positions 39 and 40 that are essential for AKAP79(31-52) peptide inhibition of PKCbetaII. Likewise, the same mutations in the full-length anchoring protein reduced inhibition of PKCbetaII. Thus AKAP79 associates with multiple PKC isoforms through a mechanism involving protein-protein interactions at the catalytic core where binding of the anchoring protein inhibits kinase activity through displacement of the pseudosubstrate.

Protein kinase C is processed by three phosphorylation events before it is competent to respond to second messengers. Specifically, the enzyme is first phosphorylated at the activation loop by another kinase, followed by two ordered autophosphorylations at the carboxyl terminus (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This study examines the role of negative charge at the first conserved carboxyl-terminal phosphorylation position, Thr-641, in regulating the function and subcellular localization of protein kinase C betaII. Mutation of this residue to Ala results in compensating phosphorylations at adjacent sites, so that a triple Ala mutant was required to address the function of phosphate at Thr-641. Biochemical and immunolocalization analyses of phosphorylation site mutants reveal that negative charge at this position is required for the following: 1) to process catalytically competent protein kinase C; 2) to allow autophosphorylation of Ser-660; 3) for cytosolic localization of protein kinase C; and 4) to permit phorbol ester-dependent membrane translocation. Thus, phosphorylation of Thr-641 in protein kinase C betaII is essential for both the catalytic function and correct subcellular localization of protein kinase C. The conservation of this residue in every protein kinase C isozyme, as well as other members of the kinase superfamily such as protein kinase A, suggests that carboxyl-terminal phosphorylation serves as a key molecular switch for defining kinase function.

The A kinase-anchoring protein AKAP79 coordinates the location of the cAMP-dependent protein kinase (protein kinase A), calcineurin, and protein kinase C (PKC) at the postsynaptic densities in neurons. Individual enzymes in the AKAP79 signaling complex are regulated by distinct second messenger signals; however, both PKC and calcineurin are inhibited when associated with the anchoring protein, suggesting that additional regulatory signals must be required to release active enzyme. This report focuses on the regulation of AKAP79PKC interaction by calmodulin. AKAP79 binds calmodulin with high affinity (KD of 28 6 4n M ( n 53)) in aC a 2 1 -dependent manner. Immunofluorescence staining shows that both proteins exhibit overlapping staining patterns in cultured hippocampal neurons. Calmodulin reversed the inhibition of PKCbII by the AKAP79(31‐52) peptide and reduced inhibition by the full-length AKAP79 protein. The effect of calmodulin on inhibition of a constitutively active PKC fragment by the AKAP79(31‐52) peptide was shown to be partially dependent on Ca 21 .C a 2 1 /calmodulin reduced PKC coimmunoprecipitated with AKAP79 and resulted in a 2.6 6 0.5-fold (n 5 6) increase in PKC activity in a preparation of postsynaptic densities. Collectively, these findings suggest that Ca 21 /calmodulin competes with PKC for binding to AKAP79, releasing the inhibited kinase from its association with the anchoring protein.

BackgroundTheadenomatous polyposis coli(APC) tumour suppressor gene encodes a 2843 residue (310 kDa) protein. APC is a multifunctional protein involved in the regulation of β-catenin/Wnt signalling, cytoskeletal dynamics and cell adhesion.APCmutations occur in most colorectal cancers and typically result in truncation of the C-terminal half of the protein.ResultsIn order to investigate the biophysical properties of APC, we have generated a set of monoclonal antibodies which enable purification of recombinant forms of APC. Here we describe the characterisation of these anti-APC monoclonal antibodies (APC-NT) that specifically recognise endogenous APC both in solution and in fixed cells. Full-length APC(1–2843) and cancer-associated, truncated APC proteins, APC(1–1638) and APC(1–1311) were produced in Sf9 insect cells.ConclusionsRecombinant APC proteins were purified using a two-step affinity approach using our APC-NT antibodies. The purification of APC proteins provides the basis for detailed structure/function analyses of full-length, cancer-truncated and endogenous forms of the protein.

One important regulatory mechanism in the control of phosphorylation events is the subcellular location of phosphatases or kinases. Several serine/threonine phosphatases and kinases have now been shown to be associated with targeting subunits; these contribute to the organization and specificity of signal transduction pathways by favoring the accessibility of their enzymes to certain substrates.

Total nuclear charge-changing cross sections σΔz in a CH2 target were measured for 54 isotopically identified secondary beams around A = 60 produced from a 1A·GeV86Kr beam via projectile fragmentation. With secondary beams between T z = 1 2 and −3, the isospin dependence of σΔz was investigated systematically. Contrary to the predictions of so-called overlap formulae, the σΔz values are not a function of the projectile mass alone, but are found to depend on the projectile isospin. A comparison of the data with the predictions of the statistical abrasion model reveals that this behaviour can be understood by the combined influence of the abrasion and the ablation phase in relativistic heavy-ion collisions. On the basis of this improved understanding, previous findings of anomalously large cross sections of secondary-reaction products are discussed.

β-catenin is a member of the armadillo repeat family of proteins and has important functions in cell-cell adhesion and Wnt signalling. Different protein species of β-catenin have been shown to exist in the cell and the relative proportions of these species are altered upon stimulation of cells with Wnt-3a (Gottardi and Gumbiner, 2004). In order to determine whether posttranslational modifications (PTMs) of β-catenin underlie these different protein species, we have used 2DE separation and immunoblotting with an antibody specific for β-catenin. High-resolution separation of differentially modified species of β-catenin in 2DE required the addition of ASB-16, a zwitterionic detergent that can solubilise integral membrane proteins. ASB-16 was also necessary for focussing of other armadillo repeat proteins, such as γ-catenin and p120-catenin. 2DE using ASB-16 allowed detection of a previously unreported phosphorylation site in the transcriptionally active form of β-catenin that binds to GST-Tcf in response to Wnt signalling.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are common in both inherited and sporadic forms of colorectal cancer (CRC), and are associated with dysregulated Wnt signaling. Colon carcinoma SW480 cells restored with stable expression of wild-type APC (SW480APC cells) exhibit attenuated Wnt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW480 and SW480APC cells to examine the effects of restored APC on exosome protein expression. A salient finding of our study was the unique expression of the Wnt antagonist Dickkopf-related protein 4 (DKK4) in SW480APC, but not parental SW480 cell-derived exosomes. Upregulation of DKK4 in SW480APC cells was confirmed by semiquantitative RT-PCR, immunoblotting, and immunogold electron microscopy. Analysis of the DKK4 gene promoter by methylation-specific PCR revealed reduced methylation in SW480APC cells, while RT-PCR demonstrated the downregulation of DNMT-3a, compared to the parental cell line. Our discovery of exosome-mediated secretion of DKK4 opens up the possibility that exosomal DKK4 may be a mechanism used by epithelial colon cells to regulate Wnt signaling which is lost during CRC progression.

β-catenin is a signaling protein with diverse functions in cell adhesion and Wnt signaling. Although β-catenin has been shown to participate in many protein-protein interactions, it is not clear which combinations of β-catenin-interacting proteins form discrete complexes. We have generated a novel antibody, termed 4B3, which recognizes only a small subset of total cellular β-catenin. Affinity proteomics using 4B3, in combination with subcellular fractionation, has allowed us to define a discrete trimeric complex of β-catenin, α-catenin and the tumor suppressor APC, which forms in the cytoplasm in response to Wnt signaling. Depletion of the limiting component of this complex, APC, implicates the complex in mediating Wnt-induced changes in cell-cell adhesion. APC is also essential for N-terminal phosphorylation of β-catenin within this complex. Each component of β-catenin/APC/α-catenin complex co-exists in other protein complexes, thus use of a selective antibody for affinity proteomics has allowed us to go beyond the generation of a list of potential β-catenin-interacting proteins, and define when and where a specific complex forms.

In this brief overview we discuss the association between Wnt signaling and colon cell biology and tumorigenesis. Our current understanding of the role of Apc in the β-catenin destruction complex is compared with potential roles for Apc in cell adhesion and migration. The requirement for phosphorylation in the proteasomal-mediated degradation of β-catenin is contrasted with roles for phospho-β-catenin in the activation of transcription, cell adhesion and migration. The synergy between Myb and β-catenin regulation of transcription in crypt stem cells during Wnt signaling is discussed. Finally, potential effects of growth factor regulatory systems, Apc or truncated-Apc on crypt morphogenesis, stem cell localization and crypt fission are considered.

The reported cDNA structure of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olson et al. Proc. Natl. Acad. Sci USA, 87:2284-2288, 1990). The calculated M(r) is 107,534 whereas the estimate by SDS-PAGE is approximately 130,000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors in the cDNA sequence for non-muscle MLCK and that the NH2-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH2-terminally blocked. A CNBr peptide derived from smMLCK contains the NH2-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating that Met-1 is present. Using a limited thermolysin digest we isolated an NH2-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH2-terminal Met being blocked by acetylation. The results demonstrate that the NH2-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating Met is not removed, but modified by alpha-NH2 acetylation of the translation product.

Smooth muscle myosin light chain kinase (MLCK) is stable in the presence of Ca2+/calmodulin and does not undergo inactivation as reported for skeletal muscle MLCK (Kennelly, P.J., Starovasnik, M.A., Edelman, A.M., and Krebs, E.G. (1990) J. Biol. Chem. 265, 1742-1749). The 61-kDa tryptic fragment of smMLCK-(283-779) with the pseudosubstrate/calmodulin binding sequence deleted undergoes rapid inactivation (t1/2 = 5 min at 25 degrees C). Thermal inactivation renders the 61-kDa fragment more susceptible to cleavage by trypsin. The pseudosubstrate sequence, smMLCK-(787-807) prevents inactivation with high potency (half-maximal protective concentration, PC0.5 = 102 +/- 9 nM). The hexapeptide smMLCK-(797-802), Arg-Arg-Lys-Trp800-Gln-Lys, protected with a similar potency (PC0.5 = 73 +/- 14 nM). The four basic residues as well as Trp were important for maintaining protection by the hexapeptide smMLCK-(797-802). Substitution of Trp800 with Ala or Leu increased the PC0.5 to 500 nM. However, substitution of both aromatic residues Tyr794 and Trp800 in the longer pseudosubstrate peptide-(787-807) had little effect, indicating that with the longer peptide other multiple interactions were sufficient to stabilize the enzyme. The peptide substrate MLC-(11-23) A14,15 was also protective (PC0.5 = 380 nM) as was Mg(2+)-ATP, Mg(2+)-ADP, and Mg2+ plus adenosine. The results demonstrate that the sequence extending from 787-815 encoding the previously identified overlapping pseudosubstrate and calmodulin binding sequences also contains residues that are essential for maintaining thermal stability but these exhibit distinct structure/function relationships.

Background The APC tumour suppressor functions in several cellular processes including the regulation of β-catenin in Wnt signalling and in cell adhesion and migration. Findings In this study, we establish that in epithelial cells N-terminally phosphorylated β-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated β-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected β-catenin, GSK3β and CK1α, but not axin. The APC/phospho-β-catenin complex in cell protrusions appears to be distinct from the APC/axin/β-catenin destruction complex. GSK3β phosphorylates the APC-associated population of β-catenin, but not the cell junction population. β-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated β-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-β-catenin accumulation in cell protrusions. Conclusions We conclude that N-terminal phosphorylation of β-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-β-catenin complexes may contribute to the tumour suppressor activity of APC.

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/β-catenin pathway, we challenged the allele combinations by genetically restricting intracellular β-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/β-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/β-catenin signaling capacity similar to that in the germline of the Apcmin mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apcmin mice arise independently of intestinal tumorigenesis. Together, the present genotype–phenotype analysis suggests tissue-specific response levels for the Wnt/β-catenin pathway that regulate both physiological and pathophysiological conditions., Author Summary Germline or somatic mutations in genes are the underlying cause of many human diseases, most notably cancer. Interestingly though, even in situations where every cell of every tissue of an organism carries the same mutation (as is the case for germline mutations), some tissues are more susceptible to the development of disease over time than others. For example, in familial adenomatous polyposis (FAP), affected persons carry different germline mutations in the APC gene and are prone to developing cancers of the colon and the rectum—and, less frequently, cancers in other tissues such as stomach, liver, and bones. Here we utilize a panel of mutant mice with truncating or hypomorphic mutations in the Apc gene, resulting in different levels of activation of the Wnt/β-catenin pathway. Our results reveal that different pathophysiological outcomes depend on different permissive signaling thresholds in embryonic, intestinal, and liver tissues. Importantly, we demonstrate that reducing Wnt pathway activation by 50% is enough to prevent the manifestation of embryonic abnormalities and disease in the adult mouse. This raises the possibility of developing therapeutic strategies that modulate the activation levels of this pathway rather than trying to “repair” the mutation in the gene itself.

Cells in tissues do not exist as isolated entities but are part of the three-dimensional tissue architecture. Consequently, some aspects of cell behaviour cannot be mimicked by simple in vitro monolayer culture systems. Moreover, cell shape and behaviour is not rigid but is dynamic and can be regulated by intrinsic and extrinsic factors. For example, tumour cells in epithelium-derived cancer such as colorectal cancer often retain significant features of the colonic mucosa. However, as the tumour progresses, the morphology of the tumour cells often undergoes a transition from an epithelial morphology to a mesenchymal morphology. This transition is important as it signifies a change in the tumour phenotype to a more aggressive, invasive, and eventually metastatic phenotype. In vitro models that allow the study of this transition are needed. One such model is the LIM1863 colon carcinoma cells that normally grow as organoids but can be adapted to efficiently undergo an epithelial to mesenchymal transition that can be reversed. This system has allowed the study of the genes such as Frizzled 7 that are involved in this dynamic and reversible epithelial to mesenchymal transition.

Cells in tissues do not exist as isolated entities but are part of the three-dimensional tissue architecture. Consequently, some aspects of cell behaviour cannot be mimicked by simple in vitro monolayer culture systems. Moreover, cell shape and behaviour is not rigid but is dynamic and can be regulated by intrinsic and extrinsic factors. For example, tumour cells in epithelium-derived cancer such as colorectal cancer often retain significant features of the colonic mucosa. However, as the tumour progresses, the morphology of the tumour cells often undergoes a transition from an epithelial morphology to a mesenchymal morphology. This transition is important as it signifies a change in the tumour phenotype to a more aggressive, invasive, and eventually metastatic phenotype. In vitro models that allow the study of this transition are needed. One such model is the LIM1863 colon carcinoma cells that normally grow as organoids but can be adapted to efficiently undergo an epithelial to mesenchymal transition that can be reversed. This system has allowed the study of the genes such as Frizzled 7 that are involved in this dynamic and reversible epithelial to mesenchymal transition.

Developmental morphogenesis relies on cell transitions between epithelial and mesenchymal states. Colorectal cancer (CRC) progression can also be described as ‘morphogenesis’ as it involves epithelial-mesenchymal transition (EMT), whereby tumour cells become more invasive and metastatic. Subsequently, the disseminated tumour cells must undergo a reverse transition (MET), as the pathology of most primary tumours is re-capitulated by their established metastases. Disseminated tumour cells can remain ‘dormant’ for many years. Consequently, tumour initiation at the secondary site is the rate-limiting step in metastasis. Metastasis is governed by cell intrinsic and extrinsic (microenvironment) factors, thus much of what we know about metastasis is drawn from in vivo model systems. However, the molecular mechanisms controlling release from ‘dormancy’ are still largely unknown due to the complexity this presents for the in vivo situation. An in vitro morphogenesis culture system would present a great advantage. To this end, we have established a unique model of CRC morphogenesis, using a variant of the human cell line LIM1863 (LIM1863-Mph). In this model system LIM1863-Mph cells show plasticity between epithelial and mesenchymal states. The transitions are reversible and bear the phenotypic hallmarks of CRC morphogenesis. Importantly, we have demonstrated a pivotal role for FZD7 in these phenotype transitions, implicating Wnt signalling in orchestrating CRC morphogenesis.

Progression of colorectal cancer (CRC) involves spatial and temporal occurrences of epithelial-mesenchymal transition (EMT), whereby tumour cells acquire a more invasive and metastatic phenotype. Subsequently, the disseminated mesenchymal tumour cells must undergo a reverse transition (mesenchymal-epithelial transition, MET) at the site of metastases, as most metastases recapitulate the pathology of their corresponding primary tumours. Importantly, initiation of tumour growth at the secondary site is the rate-limiting step in metastasis. However, investigation of this dynamic reversible EMT and MET that underpins CRC morphogenesis has been hindered by a lack of suitable in vitro models. To this end, we have established a unique in vitro model of CRC morphogenesis, which we term LIM1863-Mph (morphogenetic). LIM1863-Mph cells spontaneously undergo cyclic transitions between two-dimensional monolayer (migratory, mesenchymal) and three-dimensional sphere (carcinoid, epithelial) states. Using RNAi, we demonstrate that FZD7 is necessary for MET of the monolayer cells as loss of FZD7 results in the persistence of a mesenchymal state (increased SNAI2/decreased E-cadherin). Moreover, FZD7 is also required for migration of the LIM1863-Mph monolayer cells. During development, FZD7 orchestrates either migratory or epithelialization events depending on the context. Our findings strongly implicate similar functional diversity for FZD7 during CRC morphogenesis.

Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein-protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-catenin and consequently the strength of cell-cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.

International audience; Alterations in the Wnt/APC (adenomatous polyposis coli) signalling pathway, resulting in b-catenin/T cell factor (Tcf)-dependent transcriptional gene activation, are frequently detected in familial and sporadic colon cancers. The neuropeptide neurotensin (NT) is widely distributed in the gastrointestinal tract. Its proliferative and survival effects are mediated by a G-protein coupled receptor, the NT1 receptor. NT1 receptor is not expressed in normal colon epithelial cells, but is over expressed in a number of cancer cells and tissues suggesting a link to the outgrowth of human colon cancer. Our results demonstrate that the upregulation of NT1 receptor occurring in colon cancer is the result of Wnt/APC signalling pathway activation. We first established the functionality of the Tcf response element within the NT1 receptor promoter. Consequently, we observed the activation of NT1 receptor gene by agents causing b-catenin cytosolic accumulation, as well as a strong decline of endogenous receptor when wt-APC was restored. At the cellular level, the re-establishment of wt-APC phenotype resulted in the impaired functionality of NT1 receptor, like the breakdown in NT-induced intracel-lular calcium mobilization and the loss of NT pro-invasive effect. We corroborated the Wnt/APC signalling pathway on the NT1 receptor promoter activation with human colon carcinogenesis, and showed that NT1 receptor gene activation was perfectly correlated with nuclear or cytoplasmic b-catenin localization while NT1 receptor was absent when b-catenin was localized at the cell-cell junction in early adenomas of patients with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer and loss of het-erozygosity tumours. In this report we establish a novel link in vitro between the Tcf/b-catenin pathway and NT1 receptor promoter activation.