Essential role of JunD in cell proliferation is mediated via MYC signaling in prostate cancer cells

Abstract

JunD, a member of the AP-1 family, is essential for cell proliferation in prostate cancer (PCa) cells. We recently demonstrated that JunD knock-down (KD) in PCa cells results in cell cycle arrest in G₁-phase concomitant with a decrease in cyclin D1, Ki67, and c-MYC, but an increase in p21 levels. Furthermore, the over-expression of JunD significantly increased proliferation suggesting JunD regulation of genes required for cell cycle progression. Here, employing gene expression profiling, quantitative proteomics, and validation approaches, we demonstrate that JunD KD is associated with distinct gene and protein expression patterns. Comparative integrative analysis by Ingenuity Pathway Analysis (IPA) identified 1) cell cycle control/regulation as the top canonical pathway whose members exhibited a significant decrease in their expression following JunD KD including PRDX3, PEA15, KIF2C, and CDK2, and 2) JunD dependent genes are associated with cell proliferation, with MYC as the critical downstream regulator. Conversely, JunD over-expression induced the expression of the above genes including c-MYC. We conclude that JunD is a crucial regulator of cell cycle progression and inhibiting its target genes may be an effective approach to block prostate carcinogenesis.

Keywords: Cancer initiation; Cell cycle regulation; JunD; Prostate cancer; c-MYC.

FIGURE 1.. Generation of PC3 JunD Knock-out (KO) cells by CRISPR/Cas9 genome editing.

JunD KO in PC3 cells was confirmed by Western blot analysis (insert). Cell proliferation assays were performed to measure (A) cell growth (4 days) and (B) growth curve (1–8 days) of PC3-JunD KO cells (JunD sg1/2–1) and compared with PC3 WT (control cells). C. Cell size was determined by Image J (left panel) and visualized by staining cells with DAPI (nuclei) and Phalloidin (actin filaments), right panel. The statistical analyses were performed by one-way ANOVA analyses and Tukey Multiple Comparison test. Each bar represents Mean ± SEM (n=3) *p<0.001.

FIGURE 2.. Schematic diagram showing procedures used to examine differential gene expression and protein levels following JunD knock-down in PC3 cells.

Following JunD knock-down by siRNA and verification by western blot analysis, PC3-JunD knockdown and siControl lysates were subjected to Microarray and Proteomic analysis. Differentially expressed molecules were selected based on fold change increase or decrease in their expression (p<0.05).

FIGURE 3.. Overview of microarray and proteomic pathway analyses from PC3 cells ± JunD.

A. Putative probes regulated by JunD were identified from genes upregulated/downregulated at least 2-fold in PC3 cells after JunD knockdown compared with siControl cells. P < 0.05 was considered as significant enrichment. B. Heatmap of differentially expressed genes (F.C =1.1, p<0.01). C. A RNA microarray analysis showing the top 10 downregulated genes in PC3-JunD deficient cells which are also involved in cell cycle regulation. D. Cell Cycle Control/Regulation was one of the top pathways whose members exhibited gene expression downregulation after JunD knock-down in PC3 cells, according to Ingenuity Pathway Analysis (IPA). Pathways with p-values < 0.05 were considered significantly enriched. E, F. IPA upstream pathway analysis of mass spectrometry proteomic data revealed that MYC, a known master regulator of cell cycle, is one the top upstream regulator of JunD targets. *Green and red color in F indicates a downregulation or upregulation of protein levels, respectively, as a result of MYC inhibition.

FIGURE 4.. Integrating genomics and proteomic analyses by Ingenuity Pathway analysis.

A. Venn Diagram representing pair-wise comparison of microarray and proteomics molecules that were significantly downregulated (p<0.05). The overlap represents the common molecules (115) identified by both microarray and proteomic analyses, and 75 of those genes being cell cycle-related. The top 10 down-regulated molecules for each category are listed below its respective group. B. JunD targets are involved in cell cycle regulation and are associated with MYC pathway, according to IPA.

FIGURE 5.. Validation of the expression of the integrated genes and proteins identified.

Validation of the Microarray results of selected genes from JunD knockdown by qPCR analysis of JunD, PRDX3, CDK2, CDK4, EIF1, KIF2C, and PEA15 gene expression after JunD knockdown in A. PC3 and B. DU145 cells. C. Protein levels determined by Western blot analysis D. Quantitative analysis of relative protein levels. Normalization was performed relative to the signal obtain with α-Tuburalin. Each bar represents Mean ± SEM (n=3). Astericks represent significantly different from control groups (**p<0.01, *p<0.05). Statistical significance was determined by One Way ANOVA and Duncan’s Pairwise Multiple Comparison Method.

FIGURE 6.. JQ1, a c-MYC inhibitor, suppresses JunD-mediated cell proliferation and JunD dependent genes’ protein levels.

A. DU145 cells overexpressing JunD (D1) and DU145 cells containing an empty vector (V6), pcDNA3.1 were plated and allowed to grow for 72 hrs, followed by cell counting. Statistical significance was determined using Student’s t-Test (p<0.001). B. Western blot analyses confirming JunD overexpression in D1 cells (insert) and the protein levels of JunD dependent genes. α-Tubulin was used as a loading control. C. Cells treated with JQ1 (5μM) were subjected to cell proliferation assays, and D. western blot analysis for JunD dependent genes. Statistical significance (*p<0.001) for (C) was determined by one-way ANOVA and Duncan’s Pairwise Multiple Comparison Method.

FIGURE 7.. PRDX3 is required for cell proliferation of PC3 and DU145 cells.

PC3 and DU145 cells were transfected with either control or PRDX3 siRNA to knock-down the expression of PRDX3. Bar graph shows cell proliferation after transfections with control (siControl) or PRDX3 siRNA. Each bar represents Mean ± SEM (n=3). *The Student t-test was used to determine the significant difference from respective control groups (p <0.05). Levels of PRDX3 after transfection with siControl and PRDX3 siRNA were determined by western blot analysis (insert).

FIGURE 8.. Analyzing JunD gene expression using The Cancer Genome Atlas (TCGA) Prostate Cancer samples.

A. The expression levels (RNA-seq data) of JunD in normal prostate tissues compared with human prostate cancer (primary tumors) in TCGA database. Significance was determined using one-way ANOVA. B. The expression levels of selected JunD dependent genes (MYC, PRDX3, CDK2, KIF2C, PEA15, and EIF1) were examined in primary tumor samples with high JunD levels (n=71), indicated by the red color. C. Box plot showing the percentage (%) of high JunD primary tumors that also exhibited an increase in specific JunD dependent gene expression.

FIGURE 9.. Proposed model for JunD mediated carcinogenesis in prostate cancer cells.

Schematic model indicating that JunD activates its dependent genes (JunD target genes), whose products target c-MYC which then leads to the activation of downstream targets (JunD/c-MYC target genes) that in turn induce cell proliferation of prostate cancer cells and carcinogenesis.