Oncoprotein ZNF322A transcriptionally deregulates alpha-adducin, cyclin D1 and p53 to promote tumor growth and metastasis in lung cancer

Abstract

ZNF322A encoding a classical Cys2His2 zinc finger transcription factor was previously revealed as a potential oncogene in lung cancer patients. However, the oncogenic role of ZNF322A and its underlying mechanism in lung tumorigenesis remain elusive. Here we show ZNF322A protein overexpression in 123 Asian and 74 Caucasian lung cancer patients. Multivariate Cox regression analysis indicated that ZNF322A was an independent risk factor for a poor outcome in lung cancer, corroborating the Kaplan-Meier results that patients with ZNF322A protein overexpression had significantly poorer overall survival than other patients. Overexpression of ZNF322A promoted cell proliferation and soft agar growth by prolonging cell cycle in S phase in multiple lung cell lines, including the immortalized lung cell BEAS-2B. In addition, ZNF322A overexpression enhanced cell migration and invasion, whereas knockdown of ZNF322A reduced cell growth, invasion and metastasis abilities in vitro and in vivo. Quantitative proteomic analysis revealed potential ZNF322A-regulated downstream targets, including alpha-adducin (ADD1), cyclin D1 (CCND1), and p53. Using luciferase promoter activity assay combined with site-directed mutagenesis and sequential chromatin immunoprecipitation-PCR assay, we found that ZNF322A could form a complex with c-Jun and cooperatively activate ADD1 and CCND1 but repress p53 gene transcription by recruiting differential chromatin modifiers, such as histone deacetylase 3, in an AP-1 element dependent manner. Reconstitution experiments indicated that CCND1 and p53 were important to ZNF322A-mediated promotion of cell proliferation, whereas ADD1 was necessary for ZNF322A-mediated cell migration and invasion. Our results provide compelling evidence that ZNF322A overexpression transcriptionally dysregulates genes involved in cell growth and motility therefore contributes to lung tumorigenesis and poor prognosis.

Figure 1

Clinical significance of ZNF322A overexpression in lung cancer patients. (a) mRNA expression level of ZNF322A examined in 123 lung cancer patients. Dot plot demonstrates mRNA expression of log₁₀ ratio between ZNF322A and GAPDH in tumor tissues and corresponding normal tissues. Red line indicates mean and s.e.m. (b and c) Representative immunohistochemistry images of ZNF322A, ADD1, CCND1 and p53 protein expression in tumor specimen of two lung cancer patients are shown. Patient 1 showed ZNF322A, ADD1, and CCND1 overexpression along with p53 downregulation (b), whereas patient 2 showed a reverse pattern (c). (d) Concordance analysis between mRNA and protein expression. + indicates increased mRNA expression and positive immunoreactivity, as opposed to − for reverse patterns. The percentage of the concordant group (gray columns) and discordant group (white columns) is indicated above. (e) Overall survival of lung cancer patients with ZNF322A overexpression (solid line) versus patients with ZNF322A normal expression (dotted line). P-values for comparison (a) and correlation analyzes (d) were determined using two-tailed Student's t-test and Pearson's χ² test; and for survival analyzes (e) using log-rank test.

Figure 2

ZNF322A overexpression promotes proliferation, soft agar growth, migration and invasion abilities in lung cells. (a and b) ZNF322A overexpression (over-ZNF) promoted cell proliferation in A549, H460, CL1-5, H1299 and BEAS-2B cells, whereas ZNF322A knockdown (si-ZNF) suppressed cell proliferation. (c and d) ZNF322A overexpression promoted colony formation ability of A549, H460, CL1-5, H1299 and BEAS-2B cells, whereas ZNF322A knockdown decreased colony formation ability. (e-h) ZNF322A overexpression promoted cell migration and invasion abilities in A549, H460, CL1-5, H1299 and BEAS-2B cells. (i-l) ZNF322A knockdown decreased cell migration and invasion abilities. P-values determined using two-tailed Student's t-test. Data represent mean±s.e.m. (n=3). *P<0.05; **P<0.01; ***P<0.001.

Figure 3

ZNF322A overexpression accelerates tumor growth in vivo. (a–d) H460-Luc cells transfected with empty vector (Ctrl) or ZNF322A expression vector (over-ZNF) were subcutaneously injected into severe combined immunodeficient mice. Luciferase intensity of tumor images (a) and quantitative IVIS intensity (b) were obtained on indicated days. Xenograft images (c) and quantitative weight (d) of tumor taken from mice on day 18 are shown. (e) Xenografts were subjected to tissue-immunoblotting to examine expression level of the indicated proteins. (f–i) Control (Ctrl) or ZNF322A knockdown A549-Luc cells were subcutaneously injected into BALB/c nude mice. Luciferase intensity of tumor images (f) and quantitative IVIS intensity (g) were obtained on indicated days. Xenograft images (h) and quantitative weight (i) of tumor taken from mice at day 28 are shown. (j) Xenografts were subjected to immunohistochemistry to examine expression of the indicated proteins. Data are mean±s.e.m. (n=5 mice per group). *P<0.05; **P<0.01.

Figure 4

ZNF322A overexpression accelerates tumor metastasis in vivo. (a) Representative lung images taken from mice at 4th week are shown. A549 cells transfected with empty vector (Ctrl) or ZNF322A expression vector (over-ZNF) were injected into severe combined immunodeficient mice via the tail vein. (b and c) Lung index (lung weight/body weigh) (b) and quantitative tumor nodules (c) show bigger tumor mass and more lung nodules in over-ZNF group compared with Ctrl groups. (d) Hematoxylin and eosin staining of tumor micrometastasis are shown. (Original magnification: × 100). A twofold enlarged image is also shown with tumor nodules indicated by dotted lines. (e-h) Control (Ctrl) or ZNF322A knockdown A549 cells were injected into mice via the tail vein. Representative lung images (e), lung index (f), quantitative tumor nodules (g) and heamatoxylin and eosin staining (h) are shown. Data are mean±s.e.m. (n=5 mice per group). *P<0.05; **P<0.01.

Figure 5

ZNF322A upregulates ADD1 and CCND1 expression but downregulates p53 expression at transcriptional level. (a) A549 cell transfected with empty vector (Ctrl) or ZNF322A expression vector (over-ZNF) were harvested and subjected to immunoblotting (left) and qRT–PCR (right) analyzes as indicated. (b) A549 cell transfected with si-control (si-Ctrl) or si-ZNF322A (si-ZNF) oligo were detected for protein (left) and mRNA (right) expression. (c–f) ZNF322A transcriptionally regulates downstream gene through AP-1 elements. Dual luciferase activity assays were performed using ADD1 promoter (c and d), CCND1 promoter (e) or p53 promoter (f) containing wild-type AP-1 (blue box) or mutant AP-1 element (marked with X symbol). Data represent promoter activity in A549 cells expressing si-ZNF (gray bar) compared with si-Ctrl or over-ZNF (black bar) compared with Ctrl. TSS, transcription start site. Data are mean±s.e.m. (n=3). *P<0.05; **P<0.01; ***P<0.001.

Figure 6

ZNF322A and c-Jun bind cooperatively to AP-1 elements on ADD1, CCND1 and p53 promoters. (a) Promoter map for ADD1, CCND1 and p53 genes. AP-1 elements are indicated as blue boxes and regions examined for ChIP–qPCR are marked by red arrows. (b–g) A549 cells transfected with si-ZNF322A (si-ZNF) oligo (b-d) or si-c-Jun (si-c Jun) oligo (e–g) were subjected to ChIP–qPCR analysis. (h) Re-ChIP assays were performed in A549 cells expressing control vector (white bar) or ectopically expressing ZNF322A and c-Jun (gray bar). qPCR products of the target gene relative to input are indicated on the y axis and antibodies for the proteins analyzed are on the x axis. IgG serves as a negative control. Data are mean±s.e.m. (n=3). *P<0.05; **P<0.01; ***P<0.001.

Figure 7

ZNF322A modulates the chromatin structure of ADD1, CCND1 and p53 promoter regions. (a) Promoter map for ADD1, CCND1 and p53 genes. (b and c) A549 cells transfected with ZNF322A expression vector (over-ZNF) (b) or si-ZNF322A oligo (si-ZNF) (c) were subjected to ChIP–qPCR analyzes for acetylated histone 3 (H3ac) open chromatin mark. (d) A549 cells transfected with si-ZNF oligo were subjected to ChIP–qPCR analysis for histone deacetylase 3 (HDAC3) repressive protein. Data are mean±s.e.m. (n=3). *P<0.05; ***P<0.001. (e) Schematic representation of transcriptional induction of ZNF322A at ADD1 and CCND1 and repression at p53, and subsequent promotion of cell proliferation, tumor metastasis and poor prognosis.

Figure 8

Reconstituted ZNF322A, ADD1, CCND1 and p53 expression reverses the oncogenic effects of ZNF322A in lung cancer cells. (a and b) Knockdown of CCND1 (si-CCND1) and overexpression of p53 (over-p53) reduced cell proliferation in ZNF322A overexpressing A549 and H460 cells. (c and d) Reconstitution of ZNF322A (over-ZNF) and CCND1 (over-CCND1) and knockdown of p53 (si-p53) restored cell proliferation in ZNF322A silenced (si-ZNF) A549 and H460 cells. (e–g) Knockdown of ADD1 (si-ADD1) reversed the oncogenic effects of ZNF322A on migration and invasion abilities in vitro. (h–j) Reconstituted ZNF322A (over-ZNF) and ADD1 (over-ADD1) expression restored cell migration and invasion abilities in ZNF322A silenced lung cancer cells. Cells were transfected with indicated expression vectors with or without siRNAs. P-values determined using two-tailed Student's t-test. Data are mean±s.e.m. (n=3). **P<0.01; ***P<0.001.