RABL6A, a Novel RAB-Like Protein, Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

<div><p>RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the <u>A</u>lternative <u>R</u>eading <u>F</u>rame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis.</p></div

NIAM-Deficient Mice Are Predisposed to the Development of Proliferative Lesions including B-Cell Lymphomas

<div><p>Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation <i>Tbrg1</i>) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated <i>NIAM</i> mutant (<i>NIAM^m/m</i>) mice homozygous for a β-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged <i>NIAM</i> deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or <i>NIAM^+/m</i> heterozygous animals developed lesions. In the spleen, <i>NIAM^m/m</i> mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of <i>NIAM</i> mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free <i>NIAM</i>-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, <i>NIAM</i> down-regulation <i>in vivo</i> results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAM's role in tumorigenesis and various anti-cancer pathways, including p53 signaling.</p></div

RABL6A is a novel centrosomal protein.

<p>p53−/− MEFs infected with retroviruses expressing empty vector (VEC),wild-type RABL6A (OE) or RABL6A shRNAs (kd) were stained with antibodies to RABL6A and γ-tubulin. (A) Representative confocal images show γ-tubulin (grey or red), RABL6A (green) and DAPI-stained nuclei (blue). Merged images show γ-tubulin in red with overlap between the proteins at centrosomes appearing orange to yellow. Insets magnify individual centrosomes. Scale bar, 10 µm. (B) Quantification of RABL6A-positive pixels per centrosome in cells from (A). Error bars, SEM; *, p<0.0001 as calculated using paired, two-tailed Student’s t-test. (C) Quantification of the mean weighted overlap coefficients for centrosome co-localization of γ-tubulin and RABL6A in cells from (A). Error bars, standard deviation from the mean.</p

RABL6A associates with NPM <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Endogenous complexes between RABL6A with NPM and ARF in p53−/− MEF nuclear (Nuc) and cytosolic (Cyt) fractions were identified by IP-Western blotting using control IgG versus RABL6 antibodies (right panel). Total protein levels in nuclear versus cytosolic fractions are shown (left), with phosphorylated histone H3-S10 and α-tubulin serving as fractionation controls for the nucleus and cytosol, respectively. (B) Top, <i>In vitro</i> binding of 35S-labeled <i>in vitro</i> translated (IVT) RABL6A to GST-NPM was detected by autoradiography. Bottom, levels of input GST and GST-NPM proteins indicated by Coomassie blue gel staining (arrow denotes full-length GST-NPM).</p

CIN is a consequence of RABL6A loss in fibroblasts.

<p>p53−/− MEFs expressing CON or RABL6A shRNAs (kd1 and kd2) were infected with vector (V) or human RABL6A (R, black bars) rescue viruses. (A) Representative metaphases of CON, kd1 and kd2 cells expressing empty vector. (B) Metaphases (n = 40) were counted and the percent of cells with <u>></u>80 chromosomes graphed. Data were analyzed using a Fisher’s exact test (*, p<0.0085). (C) The frequency of micronuclei formation under each indicated condition was quantified from three or more experiments with error bars representing the standard deviation from the mean. Statistical significance was determined using two-way ANOVA analysis (*, p<0.01 compared to CON; **, p<0.05 for (R) versus (V) cells).</p

RABL6A knockdown causes centrosome amplification in WT and p53−/− MEFs.

<p>(A) Representative confocal images taken under identical settings of <i>p53</i>−<i>/</i>− CON, kd1 and kd2 cells stained for centrosomes (γ-tubulin, green) and nuclei (DAPI, blue). Yellow arrows point to areas with centrosomes, white arrows indicate micronuclei. Dashed line in the kd2 image demarcates two different cells. Scale bar, 20 µm. (B) Quantification of immunofluorescence data from (A) measuring the percent of cells with <u>></u> 3 centrosomes per cell. *, p<0.05 versus CON. (C) Immunoblot showing effective mouse RABL6A depletion in p53−/− MEFs, as well as high level overexpression of human RABL6A, with γ-tubulin levels as loading control. (D and E) Quantification of immunofluorescence data for centrosome amplification (D) and multinucleation (E) in CON, kd1 and kd2 p53−/− MEFs expressing empty vector (V) or human RABL6A (R) rescue viruses. The mean values plus standard deviations were obtained from more than 3 independent experiments (*, p<0.001 compared to CON; **, p<0.01 for human RABL6A expressing cells compared to vector controls). Statistical significance for data in panels B, D and E was determined using paired, two-tailed Student’s t-test.</p

RABL6A inhibits centrosome amplification independent of ARF.

<p>(A) Immunoblot showing effective mouse RABL6A knockdown in kd1 and kd2 triple knockout (TKO) MEFs lacking <i>p53</i>, <i>Mdm2</i> and <i>ARF</i>, as well as high level expression of human RABL6A (R) versus vector (V) in CON and kd cells. (B) Quantification of immunofluorescence data for centrosome amplification in cells from (A) from three or more separate experiments. *, p<0.01 compared to CON. **, p<0.05 for human RABL6A expressing cells versus vector controls. (C) Immunoblot showing effective mouse RABL6A knockdown in kd TKO MEFs and expression of exogenous mouse ARF in CON and kd cells. (D) Quantification of immunofluorescence data for centrosome amplification in cells from (C) from three separate experiments. *, p<0.01 compared to CON. **, p<0.05 for ARF (A) expressing cells versus vector (V) controls. #, p<0.05 for kd1 and kd2 versus CON cells expressing ARF (black bars). (E) Model showing RABL6A and ARF can act separately to inhibit centrosome amplification. The dashed arrow between ARF and RABL6A indicates ARF may suppress centrosome amplification, in part, through RABL6A. Statistical significance for data in panels B and D was determined using paired, two-tailed Student’s t-test.</p

RABL6A loss leads to centrosome amplification and multinucleation via upregulation of NPM-T199 phosphorylation.

<p>(A) p53−/− MEFs expressing CON, kd1 or kd2 shRNAs were infected with vector (V) or human RABL6A (R) rescue viruses and levels of total NPM, phosphorylated NPM at T199 (P-T199-NPM), RABL6A, and GAPDH (loading control) measured by immunoblotting. The fold change in P-T199-NPM levels (normalized to total NPM) was quantified from 3 separate studies (below). *, p<0.01 compared to CON. **, p<0.05 for (R) versus (V) samples. (B) Autoradiograph of GST-NPM phosphorylation (³²P-GST-NPM) from the indicated cell lysates following immune complex kinase assay using IgG control or Cdk2 antibodies. Graph shows the fold change in phosphorylated GST-NPM upon RABL6A loss (kd1 and kd2) or human RABL6A rescue (hRABL6A) as quantified from three experiments. * and **, p<0.01 and p<0.05 for samples relative to CON. (C, D and E) CON, kd1 and kd2 p53−/− MEFs infected with vector (VEC, V), FLAG-tagged wild-type NPM (wt) or FLAG-tagged T199A NPM mutant (mt) were assayed by western blotting (C) and immunofluorescent staining for centrosome amplification (D) and multinucleation (E). Quantified data from three separate experiments show rescue of the RABL6A loss phenotype by T199A but not WT NPM expression (*, p<0.01 compared to CON; **, p<0.01 relative to V or wt samples). Statistical significance of the data for panels A, B, D and E was determined using two-way ANOVA analyses.</p

RABL6A knockdown causes multinucleation in wild-type (WT) and p53−/− MEFs.

<p>(A) Schematic of RABL6 isoforms A-D, and specificity of RABL6 shRNAs (kd1 and kd2) for RABL6A. (B) Top, Quantification of multinucleation data obtained from WT and p53−/− MEFs immunostained for α-tubulin and DAPI (nuclear stain), showing enhanced multinucleation due to RABL6A depletion. Error bars equal standard deviation from the mean from more than three separate experiments (*, p<0.001 compared to CON, as calculated using paired, two-tailed Student’s t-test). Bottom, Western blots showing effective shRNA-mediated loss of RABL6A protein in wild-type (WT) and p53−/− MEFs, with GAPDH as loading control. (C) Representative confocal images of p53−/− MEFs immunostained for α-tubulin (green) and DAPI (blue), from which data were quantified for (B). Scale bar, 20 µm.</p

<i>NIAM^m/m</i> mice are predisposed to proliferative lesions.

<p>Histopathologic analyses of multiple mouse tissues. <b>A</b>. A hemangioma (asterisk, right panel) found in the uterus of a <i>NIAM^m/m</i> mouse compared to normal uterine tissue (left panel). (H&E, 100x) <b>B</b>. Pulmonary papillary adenoma in a homozygous <i>NIAM^m/m</i> mouse (arrow, right panel). A depiction of normal lung tissue from a control mouse is shown (left panel). (H&E, 20x) <b>C</b>. Harderian gland adenoma of a <i>NIAM^m/m</i> mouse (asterisks, right panel). A representative image of a normal Harderian gland (asterisk) from a control mouse is depicted (left panel). (H&E, 40x) <b>D</b>. A representative image of a normal liver from a control mouse (left panel) versus a focus of cellular alteration (arrows) in a <i>NIAM^m/m</i> mouse (right panel). (H&E, 40x) Controls represent tissues from similarly aged wild-type (panels B and C) and <i>NIAM^+/m</i> heterozygous (panels A and D) mice, which were found to be indistinguishable in our analyses.</p