Oncogenic zinc finger protein ZNF322A promotes stem cell-like properties in lung cancer through transcriptional suppression of c-Myc expression

Abstract

ZNF322A, a C2H2 zinc finger transcription factor, is an oncoprotein in lung cancer. However, the transcription mechanisms of ZNF322A in lung cancer stem cell-like reprogramming remain elusive. By integrating our chromatin immunoprecipitation-sequencing and RNA-sequencing datasets, we identified and validated the transcriptional targets of ZNF322A, which were significantly enriched in tumorigenic functions and developmental processes. Indeed, overexpression of ZNF322A promoted self-renewal ability and increased stemness-related gene expressions in vitro and in vivo. Importantly, ZNF322A bound directly to c-Myc promoter and recruited histone deacetylase 3 to transcriptionally suppress c-Myc expression, which in turn increased mitochondrial oxidative phosphorylation and promoted cell motility, thus maintaining stem cell-like properties of lung cancer. Clinically, ZNF322A^High/c-Myc^Low expression profile was revealed as an independent indicator of poor prognosis in lung cancer patients. Our study provides the first evidence that ZNF322A-centered transcriptome promotes lung tumorigenesis and ZNF322A acts as a transcription suppressor of c-Myc to maintain lung cancer stem cell-like properties by shifting metabolism towards oxidative phosphorylation.

Fig. 1

Establishment of ZNF322A transcriptome in lung cancer by integrating ChIP-seq and RNA-seq datasets. a Distribution of ZNF322A binding sites identified by ChIP-seq. Schematic diagram illustrates the definition of the location of a binding site in relation to a transcription unit (top panel). Pie diagram shows the location of ZNF322A binding sites relative to the nearest transcription unit (lower panel). b Snapshots of the ChIP-seq binding profiles of ZNF322A at c-Myc and ACTB genes from CLC Genomics Workbench are shown. The “coverage” represents the profile of ZNF322A binding (left). ChIP-qPCR analysis confirmed the HA-tagged ZNF322A (over-ZNF) occupancy at c-Myc gene. Cells transfected with empty vector are also shown (Ctrl). Normal IgG served as negative control. ACTB served as a negative control target gene (right). c ZNF322A binding elements were obtained by de novo motifs analysis using MEME software. The potential transcription factor binding elements related to ZNF322A targeting elements were analyzed by STAMP software. d ZNF322A transcriptional target genes were revealed by overlapping genes from RNA-seq and ChIP-seq. e, f Validation of RNA-seq datasets using qRT-PCR analysis for the expression of ZNF322A positively-regulated genes (e) and ZNF322A negatively-regulated genes (f) in cells transfected with si-control (si-Ctrl) or si-ZNF322A oligos. GAPDH gene served as internal control. Data are mean ± SEM. P values were determined using two-tailed Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001). g–i GSEA analysis showed that the expression profile in si-ZNF322A lung cancer cells was significantly and negatively enriched with lung cancer expression profiles from human lung carcinoma tissue gene set [16] (g), the human lung cancer cell gene set [Wooster et al., unpublished data deposited in Oncomine database] (h), and the zfp322a silenced mouse embryonic stem cells [8] (i)

Fig. 2

Ectopic expression of ZNF322A induces self-renewal and stemness-related gene expression in lung cancer cells in vitro and in vivo. a, b In vitro tumor sphere formation assay of H460, H1299 and A549 lung cancer cells overexpressing HA-tagged ZNF322A (over-ZNF) or empty vector (Ctrl) were photographed (a) and quantified (b). Scale bar, 200 nm. c–e qRT-PCR analysis of stemness-related genes and ZNF322A expression level in Ctrl or over-ZNF H460 (c), H1299 (d) or A549 (e) lung cancer cells. f Limited cell number (500 cells) of Ctrl or over-ZNF H460 sphere cells were mixed with matrigel and subcutaneously injected into BALB/c nude mice. Tumors were photographed after 28 days of implantation. ZNF322A overexpression in H460 cells was confirmed using immunoblotting (inset). g qRT-PCR analysis of stemness-related genes and ZNF322A expression level in H460 xenograft obtained from (f). GAPDH was used as internal control. The error bars represent SEM from three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 3

ZNF322A negatively regulates c-Myc expression at transcriptional level. a–c qRT-PCR analysis of c-Myc mRNA expression level in si-ZNF or over-ZNF H460 (a), H1299 (b) and A549 (c) lung cancer cells. d–f Immunoblotting analysis of c-Myc protein expression level in si-ZNF or over-ZNF H460 (d), H1299 (e), and A549 (f) lung cancer cells. Molecular weight endogenous ZNF322A is 44 kDa and HA-tagged ZNF322A is 47 kDa. g Minimal promoter region of c-Myc (-160/ + 555) contained two putative ZNF322A top second-MEME motifs (ZNF2, −63~−35 and +484~+512). Highly conserved sequences were mutated by site-directed mutagenesis and indicated as −1, −2, +1, and +2 (red text). h–m Dual luciferase activity assays were performed using firefly luciferase reporter vectors containing −160/+555 fragments of c-Myc promoters (Myc-715) or mutant ZNF322A binding motif of c-Myc promoters (Myc-1-mut, Myc-2-mut, Myc + 1-mut and Myc + 2-mut). Data represent promoter activity in Ctrl or over-ZNF H460, H1299 and A549 cells (h–j) or in si-Ctrl or si-ZNF cells (k–m). TSS transcription start site. n–p ZNF322A recruits HDAC3 and modulates histone acetylation (Ac-H3) on c-Myc promoter region. ChIP-qPCR and sequential ChIP-qPCR analyses were performed in si-Ctrl or si-ZNF (n) or Ctrl or over-ZNF cells (o, p). qPCR products of the target c-Myc promoter region 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. The error bars represent SEM from three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 4

ZNF322A-mediated c-Myc suppression promotes metabolic reprogramming in lung cancer cells and lung cancer spheres. a–c Oxygen consumption rate (OCR) of over-ZNF cells, over-Myc cells or reconstituted cells (over-ZNF/over-Myc) or ZNF322A overexpressed spheres of H460 (a), H1299 (b), and A549 (c) was determined. d–f ATP content of over-ZNF cells, over-Myc cells or reconstituted cells (over-ZNF/over-Myc) or ZNF322A overexpressed spheres of H460 (d), H1299 (e), and A549 (f) was examined. g–i qRT-PCR analysis of metabolism-related genes in over-ZNF cells, over-Myc cells or over-ZNF/over-Myc cells or ZNF322A overexpressed spheres of H460 (g), H1299 (h), and A549 (i). The error bars represent SEM from three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001)

Fig. 5

Reconstituted c-Myc expression reverses the oncogenic effects of ZNF322A on lung cancer cell motility but not on cell proliferation. a–i c-Myc overexpression suppressed cell migration and invasion abilities in ZNF322A overexpressing (over-ZNF/over-Myc) H460 (a-c), H1299 (d-f), and A549 (g–i) lung cancer cells. Scale bar, 200 nm. j–l c-Myc overexpression did not further promote cell proliferation abilities in ZNF322A overexpressing H460 (j), H1299 (k), and A549 (l) cells (over-ZNF/over-Myc vs. over-ZNF or over-Myc). Data are mean ± SEM (*P < 0.05; **P < 0.01; ***P < 0.001 as determined by one-way ANOVA)

Fig. 6

High ZNF322A protein expression correlates with low c-Myc mRNA expression and poor outcome in lung cancer patients. a–d Genetic alterations of ZNF322A and c-Myc in lung cancer patients from TCGA datasets. RNA-seq datasets for ZNF322A and c-Myc mRNA expression from two lung adenocarcinoma datasets, TCGA Provisional (a) and TCGA Nature 2014 [21] (b), and two lung squamous cell carcinoma datasets, TCGA Provisional (c) and TCGA Nature 2012 [22] (d), were extracted from TCGA through cBioPortal, and presented as OncoPrint (upper panel) and dot plots (lower panel) to emphasize mutual exclusivity. e Concordance analysis between ZNF322A protein and c-Myc mRNA expression (+, high expression; −, low expression) accordingly to the four molecular subtypes (−/−); (+/−); (+/+) and (−/+). ZNF322A protein expression is shown before the comma followed by c-Myc mRNA expression. The inversely correlated group included patients with (+/−) and (−/+) subtypes. P values determined using Pearson χ²-test. The percentage of cases is indicated on the pie chart. f–h Overall survival (left) and disease-free survival (right) analysis using Kaplan–Meier method indicated that patients with high ZNF322A protein expression (f), low c-Myc mRNA expression (g) and high ZNF/low Myc (+/−) expression (h) had poorer survival than other patients. P values determined using log-rank test

Fig. 7

The schematic figure shows that ZNF322A promotes stem cell-like properties of lung cancer through transcriptional suppression of c-Myc expression. a In somatic lung cancer cells, ZNF322A expresses at a mediocre level so that c-Myc can be expressed. b ZNF322A expresses at high level in cancer stem-like cells, thus recruits HDAC3 to suppress c-Myc expression through targeting to ZNF322A binding motif 2 (ZNF2) on the c-Myc promoter. Moreover, ZNF322A-mediated c-Myc suppression shifts metabolism from glycolysis to mitochondrial oxidative phosphorylation and promotes cell motilities in lung cancer stem cells