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. 2014 Jan;184(1):133-40.
doi: 10.1016/j.ajpath.2013.09.024. Epub 2013 Nov 6.

Identification of the NAC1-regulated genes in ovarian cancer

Min Gao  1 , Ren-Chin Wu  2 , Alice L Herlinger  3 , Kailee Yap  4 , Jung-Won Kim  4 , Tian-Li Wang  5 , Ie-Ming Shih  6
Affiliations

Identification of the NAC1-regulated genes in ovarian cancer

Min Gao et al. Am J Pathol. 2014 Jan.

Abstract

Nucleus accumbens-associated protein 1 (NAC1), encoded by the NACC1 gene, is a transcription co-regulator that plays a multifaceted role in promoting tumorigenesis. However, the NAC1-regulated transcriptome has not been comprehensively defined. In this study, we compared the global gene expression profiles of NAC1-overexpressing SKOV3 ovarian cancer cells and NAC1-knockdown SKOV3 cells. We found that NAC1 knockdown was associated with up-regulation of apoptotic genes and down-regulation of genes involved in cell movement, proliferation, Notch signaling, and epithelial-mesenchymal transition. Among NAC1-regulated genes, FOXQ1 was further characterized because it is involved in cell motility and epithelial-mesenchymal transition. NAC1 knockdown decreased FOXQ1 expression and promoter activity. Similarly, inactivation of NAC1 by expression of a dominant-negative construct of NAC1 suppressed FOXQ1 expression. Ectopic expression of NAC1 in NACC1 null cells induced FOXQ1 expression. NAC1 knockdown resulted in decreased cell motility and invasion, whereas constitutive expression of FOXQ1 rescued motility in cells after NAC1 silencing. Moreover, in silico analysis revealed a significant co-up-regulation of NAC1 and FOXQ1 in ovarian carcinoma tissues. On the basis of transcription profiling, we report a group of NAC1-regulated genes that may participate in multiple cancer-related pathways. We further demonstrate that NAC1 is essential and sufficient for activation of FOXQ1 transcription and that the role of NAC1 in cell motility is mediated, at least in part, by FOXQ1.

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Figures

Figure 1
Figure 1
Identification of NAC1-regulated genes. A: qPCR was performed to determine the robustness of shRNA-mediated NAC1 knockdown. NAC1 mRNA level was normalized to GAPDH. B: Western blot analysis of NAC1 protein expression after shRNA-mediated NAC1 knockdown. GAPDH was used as the loading control. C: Genome-wide transcriptional array analysis identified genes differentially expressed between SKOV3 cells treated with NAC1 shRNA virus (N24, N48) and control virus (C24, C48) at 24 and 48 hours. A threshold ratio of 1.5 was used to select candidate NAC1-regulated genes. Columns represent experimental samples, and rows represent genes. The expression level of each gene in an individual specimen is shown as a pseudocolor gradient based on the normalized, relative expression level of each gene, where red indicates overexpression and blue indicates underexpression. D: Pathway analysis of genes affected by NAC1 knockdown. A heat map shows predicted trends of the six most significantly associated categories of biological function. Each square in the heat map represents a specific biological function, and related biological functions are grouped into major categories. The size of the squares reflects the log (P value) of the biological function, with larger size indicating higher statistical significance. The color of the squares is graded from orange to blue, with orange indicating a positive Z-score (increased biological function predicted) and blue a negative Z-score. Biological functions for which a direction could not be predicted based on the current Ingenuity Knowledge Base are shown in gray.
Figure 2
Figure 2
Validation of NAC1-regulated genes (A, down-regulated; B, up-regulated) by qPCR using two independent siRNAs. qPCR was performed to confirm the candidate NAC1 target genes identified by microarray analysis. SKOV3 cells were treated with two different NAC1 siRNAs, and NAC1 knockdown efficiency was validated at both mRNA and protein levels. Relative mRNA levels of candidate genes were determined using GAPDH as a reference gene for data normalization. All results are presented as means ± SD. All except two of the candidate genes could be validated (P < 0.05) by both siRNAs.
Figure 3
Figure 3
NAC1 is both required and sufficient for FOXQ1 expression. A: Western blot analysis demonstrates a decrease in FOXQ1 protein levels 48 hours after NAC1 knockdown in both SKOV3 and HeLa cells. GAPDH was used as the loading control. B: Western blot analysis shows an increase in FOXQ1 protein levels 48 hours after ectopic NAC1 expression in NACC1−/− mouse ovarian surface epithelium. GAPDH was used as a loading control. C: qPCR demonstrates that FOXQ1 expression was induced 24 hours after transfection of a NAC1 expression construct into NACC1−/− mouse fibroblasts. D: Expression of N130, the truncated form of NAC1 comprising only the BTB domain, down-regulates FOXQ1 transcript level in HeLa cells compared with noninduced cells at different time points.
Figure 4
Figure 4
NAC1 regulates FOXQ1 promoter activity. HeLa or SKOV3 cells were first transfected with two independent NAC1 siRNAs to knock down NAC1 expression. One day later, the pGL3-FOXQ1 (black bars) promoter construct or pGL3 control plasmid (white bars) was transfected into the cells. Luciferase activity was measure 24 hours after transfection of the promoter construct. FOXQ1 promoter activity in NAC1 siRNA-treated cells is significantly lower than in the control siRNA-treated cells. ∗∗P < 0.01.
Figure 5
Figure 5
Ectopic expression of FOXQ1 increases motility of NAC1-knockdown cells. A:FOXQ1 cDNA tagged with a V5 epitope was cloned into the pLenti lentiviral vector. SKOV3 cells were transfected with the pLenti/FOXQ1-V5 or control plasmid. FOXQ1 protein expression was analyzed by Western blot using an anti-V5 antibody. GAPDH was used as a loading control. B: NAC1 siRNA- or control siRNA-treated SKOV3 cells were transfected with pLenti/FOXQ1-V5 or control plasmid, and migration (B) and invasion (C) were measured in Transwell assays. Compared with control cells (siCTRL), NAC1 siRNA-treated cells have a reduced capacity to migrate or invade in both pLenti- and pLenti/FOXQ1-V5 transfected groups (P < 0.001). Ectopic FOXQ1 expression increases the migration and invasiveness of NAC1-knockdown cells. For each group, data were normalized to control siRNA-treated samples. The error bars represent one SD. ∗P < 0.05, ∗∗P < 0.01.
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