Transcription factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression

Abstract

Background: Ubiquitin-specific protease 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in various human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown.

Methods: A luciferase reporter driven by a promoter region of USP22 gene was selectively constructed to screen against a customized siRNA library targeting 89 selected transcription factors to identify potential transcription factors (TFs) that regulate USP22 expression in human non-small cell lung cancers (NSCLC). Association of identified TFs with USP22 and potential role of the TFs were validated and explored in NSCLC by biological assays and immunohistochemistry analysis.

Results: Luciferase reporter assays revealed that SP1 and activating transcription factor 3 (ATF3) inhibit USP22 transcription, while transcription factor AP-2 Alpha/Beta (TFAP2A/2B) and c-Myc promote USP22 transcription. Binding site-directed mutagenesis and chromosome immunoprecipitation (ChIP) assays validated AP2α and AP2β are novel TFs of USP22. Furthermore, overexpression of AP2A and AP2B significantly upregulates USP22 expression, and its target: Cyclin D1, concurrently enhances the proliferation, migration, and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. Moreover, AP2 protein level correlated with USP22 protein in human NSCLC tissues.

Conclusion: Our findings indicate AP2α and AP2β are important transcription factors driving USP22 gene expression to promote the progression of NSCLC, and further support USP22 as a potential biomarker and therapeutic target for lung cancer. Video Abstract.

Keywords: AP2; ATF3; Cell proliferation; Invasion; Malignancy; NSCLC; Transcriptional regulation; USP22.

Fig. 1

Characteristics of USP22 gene promoter. A Positions of USP22 promoter regions used for constructing luciferase reporters and transcriptional activity of these luciferase reporters in lung cancer H1299 cells. B Binding sites and sequences of transcription factors SP1 and AP2 in USP22 promoter region

Fig. 2

Identification of transcription factors that regulate USP22 gene expression in lung cancer cells. A Knockdown of transcription factors of TFAP2A/2B, c-Myc, NF-YA decreased the luciferase activity of USP22 promoter, while knockdown of transcription factors of SP1 and ATF3 increased the luciferase activity of USP22 promoter. B USP22 mRNA change by knockdown of TFAP2, ATF3 and SP1 in lung cancer cells; *P < 0.05, **P < 0.01, compared to the control siRNA. C Western blot analysis shows USP22 protein change upon knockdown of these transcription factors in lung cancer cells

Fig. 3

c-Myc upregulates USP22 expression in lung cancer cells. A knockdown of c-Myc by two individual gene-specific siRNA decreased USP22 mRNA; *P < 0.05, **P < 0.01 compared to the control siRNA. B Overexpression of c-Myc increased UPS22 promoter-driven luciferase activity of in both H1299 and A549 lung cancer cells; **P < 0.01 compared to the control pcDNA empty plasmid. C Overexpression of c-Myc upregulated USP22 protein in H1299 and A549 lung cancer cells and HCT116 colorectal cancer cells

Fig. 4

Knockdown of TFAP2 downregulated USP22, suppressed cell proliferation of NSCLC cells. Knockdown TFAP2A (left panel) and TFAP2B (right panel) by siRNA decreased A USP22 mRNA (*P < 0.05, **P < 0.01 compared to the control siRNA) and B USP22 protein in H1299 and A549 cells. C Knockdown of TFAP2A (left panel) and TFAP2B (right panel) decreased the proliferation of A549 and H1299 lung cancer cells; *P < 0.05, **P < 0.01 compared to the control siRNA. D The ggscatterstats diagram shows the positive association of the USP22 mRNA to TFAP2A mRNA (left panel) and TFAP2B mRNA (right panel) in human lung cancer tissues

Fig. 5

AP2 binds to USP22 promoter. A Diagram for mutated AP2 binding site (-12 to -4). B Mutation of AP2 binding site resulted in decreased luciferase reporter activity of USP22 promoter (**P < 0.01 compared to the wild-type USP22 promoter driven luciferase reporter). C ChIP assay shows compared to IgG, a mixture of anti-AP2α and AP2β antibody pulled down significantly more USP22 promoter DNA in H1299 and A549 lung cancer cells (**P < 0.01 compared to the control IgG)

Fig. 6

Overexpression of AP2 enhances malignancy of lung cancer cells. Overexpression of AP2A (left panel, shown by anti-Flag antibody) and AP2B (right panel, shown by anti-Flag antibody) increased A USP22 Cyclin D1 proteins and B Proliferation in the parental (USP22-WT) but not the USP22-knockout (-KO, −/−) H1299 and A549 cells. C Overexpression of AP2 increased the migration of USP22-WT but not USP22-KO H1299 and A549 cells. D Quantitative data of migration assays. E Overexpression of TFAP2 increased the invasion of USP22-WT but not USP22-KO H1299 and A549 cells. F Quantitative data of invasion assays; *P < 0.05, **P < 0.01 compared to the pcDNA empty plasmid

Fig. 7

Expression of AP2 is correlated with USP22 in lung cancer tissues. Representative scored IHC staining of A AP2α, B AP2β. C Representative images of USP22, AP2α, and AP2β IHC staining in the same lung cancer tissues; 0: < 1%, 1 + : 5%, 2++: 5–25%, 3+++: > 25% positively stained cancer cells. D Spearman correlation analysis shows AP2 protein (a combination of AP2α and AP2β) was positively correlated to USP22 protein in 143 NSCLC tissues