Functional characterization of CDK5RAP3 in hepatocellular carcinoma and neuronal differentiation

published_or_final_versionAnatomyDoctoralDoctor of Philosoph

CDK5RAP3 is a novel repressor of p14ARF in hepatocellular carcinoma cells.

CDK5 regulatory subunit associated protein 3 (CDK5RAP3) is a novel activator of PAK4 and processes important pro-metastatic function in hepatocarcinogenesis. However, it remains unclear if there are other mechanisms by which CDK5RAP3 promotes HCC metastasis. Here, we showed that in CDK5RAP3 stable knockdown SMMC-7721 HCC cells, p14(ARF) tumor suppressor was upregulated at protein and mRNA levels, and ectopic expression of CDK5RAP3 was found to repress the transcription of p14(ARF). Using chromatin immunoprecipitation assay, we demonstrated that CDK5RAP3 bound to p14(ARF) promoter in vivo. Furthermore, knockdown of p14(ARF) in CDK5RAP3 stable knockdown HCC cells reversed the suppression of HCC cell invasiveness mediated by knockdown of CDK5RAP3. Taken together, our findings provide the new evidence that overexpression of CDK5RAP3 promotes HCC metastasis via downregulation of p14(ARF)

Nuclear localization of CDK5RAP3 was important for the suppression of p14ARF promoter activity.

<p>(<b>a</b>) <i>S</i>chematic diagram of CDK5RAP3 mutants (<b>b</b>) Western blotting showing the expression levels of CDK5RAP3 mutants overexpressed in HepG2. Protein lysates from reporter assay were used for Western blotting probed with anti-Myc antibody. (<b>c</b>) <i>D</i>ual luciferase reporter was performed by co-transfection of CDK5RAP3 mutants with p14^ARF-luc reporter in HepG2. Results were mean of three independent experiments, with promoter activity of vector control set as 100%. *, <i>P</i><0.05 and **, <i>P</i><0.005 compared with vector control, Student’s <i>t</i>-test. (<b>d</b>) Confocal images of wild type (WT) and the indicated deletion mutants of Myc-CDK5RAP3.</p

Regulation of p14^ARF localization and protein expression by CDK5RAP3.

<p>(<b>a</b>) The p14^ARF and CDK5RAP3 protein levels in stable CDK5RAP3 knockdown SMMC-7721 clones (shCDK5RAP3#1 and #2), vector control and parental cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042210#pone.0042210-Mak1" target="_blank">[5]</a> were compared by Western blotting using indicated antibodies, respectively. (<b>b</b>) The stable CDK5RAP3 knockdown SMMC-7721 (shCDK5RAP3#2) and vector control cells were treated with 100 µg/ml cycloheximide or DMSO (vehicle), and harvested at the indicated time points. p14^ARF and <i>β</i>-actin protein levels were determined by Western blotting. V: DMSO treatment. <i>Top</i>: Western blotting; <i>bottom</i>: quantification of p14^ARF protein level.</p

Suppression of endogenous expression of p14^ARF by CDK5RAP3.

<p>(<b>a</b>) The <i>p14^ARF</i> and <i>CDK5RAP3</i> mRNA expression in stable CDK5RAP3 knockdown SMMC-7721 stable clones was determined by Quantitative real-time PCR (qPCR). Data was analyzed by comparative Ct method. Band intensity was analyzed using AlphaEasePC software and normalized with <i>β-actin</i>. Results were mean of three independent experiments. *, <i>P</i><0.005, Student’s <i>t</i>-test. (b) Similar to (a), the CDK5RAP3 stable expressing HepG2 clones (CDK5RAP3#1 and #2), vector control and parental cells were used for qPCR assay. Results were mean of three independent experiments. *<i>P</i><0.04 and **<i>P</i><0.02 compared with vector control, Student’s <i>t</i>-test. (c) The CDK5RAP3 expression construct and p14^ARF luciferase reporter, pGL3-p14^ARF were co-transfected into SMMC-7721 cells for dual-luciferase reporter assay. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.05 compared with vector control, Student’s <i>t</i>-test. (d) Similar to (c), luciferase reporters carrying truncation mutants of the p14^ARF promoter, CDK5RAP3 expression construct (0.3 µg) and vector (0.3 µg) were used for dual-luciferase reporter assay. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.02 compared with vector control, Student’s <i>t</i>-test. (e) CDK5RAP3 bound <i>p14^ARF</i> promoter by performing chromatin immunoprecipitation (ChIP) analysis on CDK5RAP3 stable overexpression clone #2 HepG2 cells (1×10⁷). Input (IN) and no antibody control (No Ab) were included. Error bars: mean ±SD.</p

Knockdown of p14^ARF reversed the suppression of cell migration and invasiveness in CDK5RAP3 knockdown HCC cells.

<p>(<b>a</b>) The CDK5RAP3 stable knockdown SMMC-7721 cells were transfected with p14^ARF or control siRNA. <i>Top,</i> Western blotting showing p14^ARF knockdown in cells; <i>bottom,</i> The bar chart showed the quantitation of migrated cells in three independent experiment (*, <i>P</i> = 0.005, Student’s <i>t</i>-test). Representative photomicrographs were shown. (<b>b</b>) Similar to <b>a</b>), but invasion assay were performed. The bar chart showed the quantitation of the invaded cells in three independent experiment (*, <i>P</i> = 0.05, Student’s <i>t</i>-test).</p

CDK5RAP3 transcriptionally regulated p14^ARF in a E2F1 independent manner.

<p>(<b>a</b>) CDK5RAP3 was co-transfected with p53-responsive element reporter for luciferase assay in HepG2 cells. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.05, **, <i>P</i><0.005 compared with vector control, Student’s <i>t-</i>test. (<b>b</b>) Expression constructs of CDK5RAP3 was co-transfected with HA-E2F1 and p14^ARF-luc reporter for luciferase assay in HepG2 cells. Results represent mean ±SD for triplicate wells. *, <i>P</i><0.005 compared with vector control, Student’s <i>t</i>-test.</p