<i>Nkd</i> mutations in fly and human.

<p>(A) Wild-type, <i>axin, apc</i>, and <i>nkd Drosophila</i> cuticles. Wild type has alternating denticle bands (arrow) and naked cuticle (arrowhead), with each mutant lacking denticle bands. (B) <i>NKD1</i> locus has 10 exons. Nkd1 schematic (orange) includes N-terminal myristoylation, EFX, 30aa (blue), and carboxy-terminal His-rich motifs. Exon 10 sequences around poly-(C) tracts (red) above native (black) and mutant (blue) residues are shown. (C) <i>NKD1</i> electropherograms showing wild-type (WT) poly-(C)₇, cell line TC7 with C-deletion (C6), cell line RKO with C-insertion (C8), and cell line HCT15 with G>A mutation (arrow) 3′ of poly-(C)₇. (D, E) α-Nkd1 blots of whole cell extracts (D) and Triton X-100 soluble and insoluble fractions (E) from cell lines with indicated <i>NKD1</i> mutation. Arrows designate Nkd1 proteins. β-actin is loading control in D. CCD841 has full length Nkd1, with a minor degradation product at ∼35 kDa also seen with transfected <i>NKD1</i> (e.g. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007982#pone-0007982-g001" target="_blank">Fig. 1E</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007982#pone-0007982-g005" target="_blank">5C</a>), whereas SW480 with more abundant but wild-type Nkd1 has several degradation products.</p

Effect of <i>NKD1</i>-mutations on β-catenin and cell proliferation.

<p>(A) Western blot of cytosolic and nuclear extracts of cells with wild-type (CCD841) or mutant (Co115) <i>NKD1</i>. GAPDH and HDAC2 were probed as loading controls. (B–B″, C–C″) β-catenin (red) and DNA (blue) distribution in CCD841 cells (B–B″) and Co115 (C–C″) cells. Arrows designate nuclei. Merged images in B″ and C″. (D) Western blot of cytosolic extracts of HEK-293 cells transfected with <i>lacZ</i> control (-), wild type Nkd1 (WT), or indicated mutant Nkd1 construct, and probed for β-catenin, Nkd1, and loading control GAPDH. Note that each mutant Nkd1 but not wild type Nkd1 increases β-catenin levels. (E) Relative cell number as a function of days post retroviral infection of CCD841 cells with empty vector control, wild type Nkd1, or indicated Nkd1 mutant (p = 0.016, 0.012, and 0.0091 for C6, C8, and R288H mutants as compared to control) (F) Relative cell number as a function of days post retroviral infection of Co115 cells with control or wild-type Nkd1 (p = 0.022). α-Nkd1 western blots of cell extracts, with GAPDH or β-actin loading control, are shown below each plot in E and F.</p

Mutant Nkd1s do not limit the abundance of Dsh/Dvl as well as wild type Nkd1.

<p>(A–A″) <i>71B-Gal4/UAS-Nkd^GFP</i> third-instar <i>Drosophila</i> salivary gland stained with α-Dsh and imaged for GFP (A) and Dsh (A′) distributions (merged image in A″). Quantitation of Dsh pixel intensity (white box in A′) reveals reduced staining in cell expressing more (right, green asterisk) Nkd^GFP than in adjacent cell expressing less Nkd^GFP. (B) Western blots of HEK-293 cells transfected with indicated Flag/HA-tagged Nkd1 and Dvl1, Dvl2, or Dvl3 constructs probed with α-HA, α-Dvl1-3, and α-βtubulin as a loading control. Each of the Dvl1-3 blots was loaded with equal amounts of extract, as confirmed by probing each blot with α-βtubulin. (C) HEK-293 cells co-expressing Nkd1^GFP and Dvl1, stained with α-Dvl1, and imaged for GFP (green), Dvl1 (red), and DNA (blue) showing fine punctate Dvl1 distribution in cells expressing low to absent Nkd1^GFP (white arrow). In adjacent cells expressing Nkd1^GFP, Dvl1 is relocalized to Nkd1^GFP/Dvl1 aggregates (yellow arrow), with loss of fine punctate Dvl1 staining (arrowheads). (D, E) Models of Nkd function in <i>Drosophila</i> (D) and <i>NKD1</i>-mutant CRC (E). Double lines: upper = plasma membrane; lower = nuclear membrane. (D) Fly Wg(Wnt) binds Fz/Arrow(Lrp5/6) receptors, which inhibits the Apc/Axin/CK1/GSK3β complex that promotes degradation of Arm(β-catenin). Arm complexes with Pan(TCF) to activate target genes including <i>nkd</i>. Nkd promotes Dsh turnover to partially inhibit signaling, and employs the nuclear import factor Imp-α3 to enter the nucleus and further inhibit signaling through unknown mechanisms <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007982#pone.0007982-Chan3" target="_blank">[31]</a>. (E) In <i>NKD1</i>-mutant CRC, the mutant Nkd1 protein no longer binds and promotes Dvl turnover, stabilizing β-catenin and activating TCF-dependent transcription of target genes.</p

Wnt-pathway genetic lesions in MSI colon tumors and cell lines.

<p><i>NKD1, AXIN2, TCF7L2, CTNNB1</i>, and <i>APC</i> gene mutation status in 40 MSI-CRC tumors (top) and 11 cell lines (bottom). Key: −, no lesion; ins, nucleotide insertion; del, nucleotide deletion; /−, allelic deletion. For <i>NKD1</i> and <i>AXIN2</i>, mutation in indicated nucleotide is designated, while for <i>TCF7L2</i> the status of the poly(A) tract (A8–wild-type; A9–mutant) is designated. <i>CTNNB1</i> exon-3 was screened for activating mutations (M). For <i>APC</i>, tumors positive for protein-truncation (T) are indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007982#pone.0007982-Powell1" target="_blank">[45]</a>. Presence (+) or absence (−) of <i>APC</i> and <i>CTNNB1</i> lesions in each cell line is as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007982#pone.0007982-Gayet1" target="_blank">[48]</a>.</p

Nkd1 truncation alters subcellular localization and Dvl colocalization.

<p>(A, B) HEK-293 cells expressing HA/Flag-tagged Nkd1 (A) or Nkd1^C8 (B) stained with α-HA (green). Nkd1 accumulates in puncta (arrows), while Nkd1^C8 is diffusely distributed in the cytoplasm. Nkd1^C6 distributed in a pattern similar to Nkd1^C8 (not shown). (C, D) Cells co-expressing Dvl3 and wild-type (C) or C8 (D) Nkd1^GFP constructs. Nkd1-GFP (green, C) shows near complete colocalization (arrows) with Dvl3 (red, C′), while Nkd1^C8-GFP (D) accumulates in cytoplasmic aggregates surrounded by Dvl3 (arrows). Merged images are in C″, D″, DNA is blue in A–D. Similar results were observed in cells coexpressing Nkd1^C8-GFP and Dvl1 or Dvl2, and in cells expressing Nkd1^C6-GFP and Dvl1, Dvl2, or Dvl3 (not shown). (E) Salivary gland from wild-type third instar <i>Drosophila</i> larva stained with α-Dsh (red) showing diffuse and punctate staining. (F–F″) <i>A8-Gal4/UAS-Nkd^GFP</i> salivary gland stained with α-Dsh and imaged for GFP (F) and Dsh (F′; merged in F″). Fly Nkd^GFP relocalizes Dsh to perinuclear aggregates (arrows) similar to those observed with Nkd1^GFP/Dvl3 in C.</p

Wild-type Nkd1 but not mutant Nkd1 inhibits Wnt/β-catenin signaling.

<p>(A) % <i>Xenopus</i> embryos with indicated axis phenotype after injection of <i>XWnt8</i>+/−wild-type or mutant <i>NKD1</i>. (n) = # embryos injected. Panels at right show representative lateral (L) and dorsal (D) views of embryos scored as single axis (white), short A/P axis (light gray), partial axis duplication (dark gray), and full axis duplication (black) according to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007982#s4" target="_blank">Materials and Methods</a>. In embryos with single axis in the left panels, note trunk (white arrow) and cement gland (black arrow). Arrowheads designate each axis in embryos with axis duplications (right panels). Embryos with partial axis duplication have duplicated trunk tissue but a single cement gland, while embryos with full axis duplication have duplicated trunk tissues and cement glands. (B) Normalized TOPflash luciferase activity (RLU) in HEK-293 cells transfected with reporter +/−Dvl2 +/− indicated Nkd1 construct.</p

Correlation of Chromosomal Instability, Telomere Length and Telomere Maintenance in Microsatellite Stable Rectal Cancer: A Molecular Subclass of Rectal Cancer

<div><p>Introduction</p><p>Colorectal cancer (CRC) tumor DNA is characterized by chromosomal damage termed chromosomal instability (CIN) and excessively shortened telomeres. Up to 80% of CRC is microsatellite stable (MSS) and is historically considered to be chromosomally unstable (CIN+). However, tumor phenotyping depicts some MSS CRC with little or no genetic changes, thus being chromosomally stable (CIN-). MSS CIN- tumors have not been assessed for telomere attrition. </p> <p>Experimental Design</p><p>MSS rectal cancers from patients ≤50 years old with Stage II (B2 or higher) or Stage III disease were assessed for CIN, telomere length and telomere maintenance mechanism (telomerase activation [TA]; alternative lengthening of telomeres [ALT]). Relative telomere length was measured by qPCR in somatic epithelial and cancer DNA. TA was measured with the TRAPeze assay, and tumors were evaluated for the presence of C-circles indicative of ALT. p53 mutation status was assessed in all available samples. DNA copy number changes were evaluated with Spectral Genomics aCGH. </p> <p>Results</p><p>Tumors were classified as chromosomally stable (CIN-) and chromosomally instable (CIN+) by degree of DNA copy number changes. CIN- tumors (35%; n=6) had fewer copy number changes (<17% of their clones with DNA copy number changes) than CIN+ tumors (65%; n=13) which had high levels of copy number changes in 20% to 49% of clones. Telomere lengths were longer in CIN- compared to CIN+ tumors (p=0.0066) and in those in which telomerase was not activated (p=0.004). Tumors exhibiting activation of telomerase had shorter tumor telomeres (p=0.0040); and tended to be CIN+ (p=0.0949).</p> <p>Conclusions</p><p>MSS rectal cancer appears to represent a heterogeneous group of tumors that may be categorized both on the basis of CIN status and telomere maintenance mechanism. MSS CIN- rectal cancers appear to have longer telomeres than those of MSS CIN+ rectal cancers and to utilize ALT rather than activation of telomerase. </p> </div

Tumor versus adjacent normal gene expression profiles of the <i>cis</i>-eQTL associated genes.

<p>Box plots of gene expression levels for <i>ATP5C1</i>, <i>DLGAP5</i>, <i>NOL3</i>, <i>and DDX28</i> in paired adjacent normal colon tissue and colon tumor tissue (n = 40 pairs). The significance of differential expression is indicated by the <i>p</i>-value.</p

Expression of four genes found to differ by genotype for three colorectal cancer risk variants.

<p>Box plots of normalized gene expression levels of <i>ATP5C1</i>, <i>DLGAP5</i>, <i>NOL3</i>, <i>and DDX28</i> for paired adjacent normal colon tissue (n = 40) and colon tumor tissue (n = 40). Each point represents the normalized RNA expression levels for an individual. The median gene expression level for each genotype specific group is indicated by a line inside each box within the graph. The <i>p</i>-value indicates the significance of the global test comparing expression across genotypes. If the p-values were significant (<i>p</i>-value≤0.05), the FDR <i>q</i>-values were provided, indicating the significance after correction for multiple comparisons.</p

Nineteen established CRC risk variants identified by GWAS and their proxies considered in this study.

a<p>Position based on dbSNP build 130.</p>b<p>Major allele/minor allele among Europeans.</p>c<p>Minor allele frequencies from published reports.</p>d<p>Linkage disequilibrium between SNP and proxy in HapMap CEU.</p>e<p>Not on Affymetrix 6.0 array.</p>f<p>Excluded from analysis as proxy r²<0.90.</p