NANOG regulates glioma stem cells and is essential in vivo acting in a cross‐functional network with GLI1 and p53

A cohort of genes associated with embryonic stem (ES) cell behaviour, including NANOG, are expressed in a number of human cancers. They form an ES‐like signature we first described in glioblastoma multiforme (GBM), a highly invasive and incurable brain tumour. We have also shown that HEDGEHOG‐GLI (HH‐GLI) signalling is required for GBM growth, stem cell expansion and the expression of this (ES)‐like stemness signature. Here, we address the function of NANOG in human GBMs and its relationship with HH‐GLI activity. We find that NANOG modulates gliomasphere clonogenicity, CD133⁺ stem cell cell behavior and proliferation, and is regulated by HH‐GLI signalling. However, GLI1 also requires NANOG activity forming a positive loop, which is negatively controlled by p53 and vice versa. NANOG is essential for GBM tumourigenicity in orthotopic xenografts and it is epistatic to HH‐GLI activity. Our data establish NANOG as a novel HH‐GLI mediator essential for GBMs. We propose that this function is conserved and that tumour growth and stem cell behaviour rely on the status of a functional GLI1‐NANOG‐p53 network.     

Grb2 and Gads exhibit different interactions with CD28 and play distinct roles in CD28-mediated costimulation

Although both CD28 and ICOS bind PI3K and provide stimulatory signal for T cell activation, unlike CD28, ICOS does not costimulate IL-2 secretion. CD28 binds both PI3K and Grb2, whereas ICOS binds only PI3K. We have generated an ICOS mutant, which can bind Grb2 by replacement of its PI3K binding motif YMFM with the CD28 YMNM motif, and shown that it induces significant activation of the IL-2 promoter. However, this mutant ICOS was insufficient to activate the NF-kappaB pathway. In this study, we show that Gads, but not Grb2, is essential for CD28-mediated NF-kappaB activation, and its binding to CD28 requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. Mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduce their association with Gads, whereas their associations with Grb2 are maintained. They induced strong activity of the NFAT/AP-1 reporter comparable with the CD28 wild type, but weak activity of the NF-kappaB reporter. Grb2- and Gads-dominant-negative mutants had a strong effect on NFAT/AP-1 reporter, but only Gads-dominant-negative significantly inhibited NF-kappaB reporter. Our data suggest that, in addition to the PI3K binding motif, the PXXP motif in the CD28 cytoplasmic domain may also define a functional difference between the CD28- and ICOS-mediated costimulatory signals by binding to Gads.     

Tethering of apoptotic cells to phagocytes through binding of CD47 to Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1

Apoptotic cells are swiftly phagocytosed by macrophages and immature dendritic cells. In this study, we found that one mouse macrophage cell line (BAM3) engulfed apoptotic thymocytes, but not a lymphoma cell line (WR19L). mAbs that inhibited the phagocytosis of apoptotic thymocytes by BAM3 were identified. Purification of the Ag revealed that it was Src homology 2 domain-bearing protein tyrosine phosphatase substrate-1 (SHPS-1). CD47, the ligand for SHPS-1, was expressed in mouse thymocytes, but not in WR19L. When WR19L was transformed with CD47, the transformants, after induction of apoptosis, could be phagocytosed by BAM3. The WR19L transformants expressing CD47 were more efficiently engulfed in vivo by splenic dendritic cells than the parental WR19L. Masking of the phosphatidylserine exposed on apoptotic thymocytes inhibited the engulfment, whereas the anti-SHPS-1 mAb inhibited not only the engulfment, but also the binding of apoptotic cells to phagocytes. These results indicate that macrophages require CD47 and phosphatidylserine on apoptotic cells for engulfment, and suggest that the interaction between CD47 and SHPS-1 works as a tethering step in the phagocytosis.     

NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling, and differentiation is promoted by BMP signaling, but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium, we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG, OCT4, and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal.     

MiR‐30‐5p suppresses cell chemoresistance and stemness in colorectal cancer through USP22/Wnt/β‐catenin signaling axis

Colorectal cancer (CRC) remains both common and fatal, and its successful treatment is greatly limited by the development of stem cell‐like characteristics (stemness) and chemoresistance. MiR‐30‐5p has been shown to function as a tumor suppressor by targeting the Wnt/β‐catenin signaling pathway, but its activity in CRC has never been assessed. We hypothesized that miR‐30‐5p exerts anti‐oncogenic effects in CRC by regulating the USP22/Wnt/β‐catenin signaling axis. In the present study, we demonstrate that tissues from CRC patients and human CRC cell lines show significantly decreased miR‐30‐5p family expression. After identifying the 3’UTR of USP22 as a potential binding site of miR‐30‐5p, we constructed a luciferase reporter containing the potential miR‐30‐5p binding site and measured the effects on USP22 expression. Western blot assays showed that miR‐30‐5p decreased USP22 protein expression in HEK293 and Caco2 CRC cells. To evaluate the effects of miR‐30‐5p on CRC cell stemness, we isolated CD133 + CRC cells (Caco2 and HCT15). We then determined that, while miR‐30‐5p is normally decreased in CD133 + CRC cells, miR‐30‐5p overexpression significantly reduces expression of stem cell markers CD133 and Sox2, sphere formation, and cell proliferation. Similarly, we found that miR‐30‐5p expression is normally reduced in 5‐fluorouracil (5‐FU) resistant CRC cells, whereas miR‐30‐5p overexpression in 5‐FU resistant cells reduces sphere formation and cell viability. Inhibition of miR‐30‐5p reversed the process. Finally, we determined that miR‐30‐5p attenuates the expression of Wnt/β‐catenin signaling target genes (Axin2 and MYC), Wnt luciferase activity, and β‐catenin protein levels in CRC stem cells.     

Origins of PDZ domain ligand specificity. Structure determination and mutagenesis of the Erbin PDZ domain

The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW(-4)ETW(-1)V; IC(50) = approximately 0.15 microm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp(-1) are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the beta2-beta3 loop provides a binding site for the Trp(-4) side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.     

Studies on the structure and binding properties of the cysteine-rich domain of rat low affinity nerve growth factor receptor (p75NGFR).

The binding domain of p75NGFR contains four "repeats" of a 6-cysteine pattern. To test whether these repeats have any structural or functional independence, each repeat has been separately deleted. In each case, deletion led to the loss of most nerve growth factor (NGF) binding activity. The epitopes of two monoclonal antibodies, MC192 and 271c, could be distinguished, however. Repeat IV was found to be unnecessary for binding MC192, whereas Repeat I was not required for binding 217c. This suggests that either terminal repeat can be removed without loss of native-like structure in the remaining repeats. Trp155 in the fourth repeat forms an essential part of the 217c epitope but is not required for either MC192 or NGF binding. Deletion of the linker region between the membrane-spanning domain and the cysteine-rich domain does not affect the binding of NGF or MC192, and has only a slight, if any, effect on 217c binding. Cyclic permutation of the four repeats failed to yield protein capable of binding NGF or MC192.     

Structural consequences of divalent metal binding by the adenylyl cyclase toxin of Bordetella pertussis.

Adenylyl cyclase toxin of Bordetella pertussis has been shown by several investigators to require Ca(2+) for its actions on target cells, but little is known about the nature and specificity of divalent metal binding to this novel toxin. Calcium is the preferred divalent metal since toxic actions are markedly reduced in the presence of divalent species other than calcium. Mn(2+) EPR was used to quantitate and characterize divalent metal binding and revealed that the toxin contains approximately 40 divalent metal sites, consisting of at least one class of high-affinity sites that bind Mn(2+) with a K(D) of 0.05 to 0.35 microM and one or more classes of lower affinity sites. Water proton relaxation data indicate that approximately 30 of these sites are completely inaccessible to bulk solvent. Our observations, together with the sequence homology between adenylyl cyclase toxin and the alkaline protease of Pseudomonas aeruginosa, indicate that the formation of five beta-sheet helices within the repeat domain of the toxin upon binding Ca(2+) is required for cell intoxication.