Transcription factors with binding sites over-represented in promoters of genes upregulated by K13 in BCBL1 and HUVECs.

<p>(Genes upregulated more than 1.5-fold were analyzed using the JASPAR and TRANSFEC databases. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037498#s3" target="_blank">Results</a> with a p-value of less than 0.05 are shown. % input refers to the number of gene promoters bearing the specific motif compared to total number screened.</p

Gene set enrichment analysis.

<p>For Gene set enrichment analysis of signatures genes from BCBL1-K13 (top panel) and HUVEC-K13 (lower panel), the t-test was graphed for each correlated gene in the ranked dataset. Three GSEA enrichment plots for representative biological pathways (Cytokine, NF-κB and Inflammatory) enriched in 4OHT-treated BCBL1-K13-ER^TAM and HUVEC-K13-ER^TAM are shown. The top portion of each GSEA plot shows the running enrichment score for validated genes specific for particular pathway as it moves down the ranked list. The bottom portion of each plot shows the value of ranking matrices as it moves down the list of ranked genes. The red horizontal bar which terminate with blue color indicate shift from positively correlated genes (red) to negatively correlated genes (blue). Further detailed interpretation about these plots can be found at Broad Institute web site (<a href="http://www.broadinstitute.org/gsea/index.jsp" target="_blank">http://www.broadinstitute.org/gsea/index.jsp</a>).</p

Summary of differentially regulated gene clusters in 4OHT-treated K13-ER^TAM-transduced BCBL1 cells.

<p>Summary of differentially regulated gene clusters in 4OHT-treated K13-ER^TAM-transduced BCBL1 cells.</p

Validation of gene array data by qRT-PCR.

<p>(A) Twenty five genes from NF-κB, cytokine, and inflammatory pathways were randomly selected and their relative mRNA levels in mock and 4OHT-treated vector and K13-ER^TAM-expressing BCBL1 cells were examined using qRT-PCR. Real-time PCR reactions were performed in triplicate and the data presented as fold change mean ±S.E in target gene expression (*p<0.05; Student's t-test). (B) Pearson Correlation coefficient between gene expression array and real time PCR showed a significant agreement (Correlation coefficient 0.88; p<0.0001).</p

NF-κB inhibitors block K13-regulated genes.

<p>BCBL1 K13-ER^TAM cells were treated with two NF–κB inhibitors (2 µM Bay 11-7082 or 2 µM As2O3) for 2 hours followed by 4OHT treatment for additional 24 hours and total RNA was isolated as described in the Materials and Method section. Nine genes were randomly picked and their relative mRNA levels were examined using real-time RT-PCR as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037498#pone-0037498-g002" target="_blank">Figure 2A</a>.</p

Gene sets enriched in BCBL-K13 and HUVEC-K13 cells (a) Biocarta gene sets, (b) KEGG (Kyoto Encyclopedia of Genes and Genomes) gene sets.

<p>Gene sets enriched in BCBL-K13 and HUVEC-K13 cells (a) Biocarta gene sets, (b) KEGG (Kyoto Encyclopedia of Genes and Genomes) gene sets.</p

Starvation- and p53-induced degradation of TBC1D15.

<p>(A) Flag- TBC1D15 was expressed alone or with myc-p53 as indicated, followed by cell lysis and analysis by immunoblotting. (B) p53 transactivation function mediates destabilization of myc-TBC1D15. HEK-293A cells were transfected with myc-TBC1D15 and either empty vector or Flag-p53 (human), wild-type murine p53 or the transactivation-deficient point mutant p53D278N, followed by lysis and immunoblotting. (C) Cells were transfected with Flag-TBC1D15 and either empty vector or myc-p53, followed by exposure for 6 hr to autophagy inhibitor Bafilomycin A1 (100 nM) or proteasome inhibitor lactacystin (5 μM). (D) HEK-293A cells expressing myc-TBC1D15 were exposed for 6 hr to DMSO vehicle or to the mTOR inhibitors rapamycin (100 nM) or PP242 (1 μM), then lysed and analyzed by immunoblotting. (E) Cells were transfected with myc-TBC1D15 and cultured in complete media or shifted to glucose- and amino acid-free media for 6 hr in the absence or presence of Bafilomycin A1, as indicated. (F) Elevated levels of TBC1D15 in <i>Atg5</i>- and <i>p53</i>-deficient livers. Livers were surgically resected from p53-deficient mice or from mice with hepatocyte-specific deletion of <i>Atg5</i> (<i>Atg5^fl</i>^/fl; <i>Alb-cre</i>) or littermate controls. Liver lysates were resolved by SDS-PAGE and analyzed by immunoblotting.</p

The TBC1D15 Oncoprotein Controls Stem Cell Self-Renewal through Destabilization of the Numb-p53 Complex

<div><p>Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 tumor suppressor, in conjunction with its interacting partner protein Numb, preserves this asymmetry and functions as a vital barrier against the unchecked expansion of tumor stem cell pools; however, little is known about the biological control of the Numb-p53 interaction. We show here that Numb and p53 are the constituents of a high molecular mass complex, which is disintegrated upon activation of aPKCζ, a Numb kinase. Using large-scale affinity purification and tandem mass spectrometry, we identify TBC1D15 as a Numb-associated protein and demonstrate that its amino-terminal domain disengages p53 from Numb, triggering p53 proteolysis and promoting self-renewal and pluripotency. Cellular levels of TBC1D15 are diminished upon acute nutrient deprivation through autophagy-mediated degradation, indicating that TBC1D15 serves as a conduit through which cellular metabolic status is linked to self-renewal. The profound deregulation of TBC1D15 expression exhibited in a diverse array of patient tumors underscores its proposed function as an oncoprotein.</p> </div

Conceptual model of TBC1D15 function in TISC-mediated oncogenesis.

<p>The transition from an untransformed hepatocyte to a TISC is accompanied by an anabolic shift and increased levels of the TBC1D15 oncoprotein, which destabilizes the Numb-p53 complex to promote deregulated self-renewal and oncogenesis.</p

Oncogenic function of TBC1D15.

<p>(A) Tumor initiation titration. Defined numbers of TISCs stably expressing the indicated transgenes or lentivirus shRNAs were implanted subcutaneously into the dorsal hind flanks of NOG mice and tumor growth monitored for 60 days. Tumors greater than 25 mm³ and which exhibited growth progression during the course of the study were scored as positive. (B) Tumor growth kinetics. TISCs (5× 10⁴) were implanted subcutaneously into NOG mice as in (A) and tumor volumes were measured on the indicated days. At least 6 tumors were measured for each cell line examined. (C) TBC1D15 expression in HCC patient tissue specimens. Tumors and matched, non-cancerous control tissues were processed by sectioning and immunostained using TBC1D15 antibody, then counterstained with hematoxylin to indicate cell nuclei. Below, high magnification image showing punctate localization of TBC1D15 (arrows) in noncancerous control tissue. Scale bar, 50 μm. (D) Stained tissues from patient specimens were scored for the degree of TBC1D15 immunopositivity. Brackets indicate statistically significant (**<i>P</i><0.01) comparisons between groups. The number of total cells scored across all samples (n = 17) is indicated for each group. (E) Box plot showing the expression of TBC1D15 in diverse tumor types and matched normal tissues. Red line indicates the median, box edges the 25–75 percentiles. Whiskers represent 1.5×interquartile range (IQR) above the third quartile or below the first quartile. Outliers (blue circles or black triangles) are shown when outside this range. (F) Scatter plots showing results from meta-analysis comparing the expression levels of <i>TBC1D15</i> and <i>NANOG</i> across multiple tumor samples and in matched, non-tumor control tissues.</p