Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma

Abstract

Deregulated monoubiquitination of histone H2B (H2Bub1), mainly catalyzed by E3 ubiquitin-protein ligase RNF20/RNF40 complex, may play an important role in cancer. Here we investigate potential roles of H2Bub1 and the underlying mechanisms through which it contributes to cancer development and progression in lung adenocarcinoma. We show that downregulation of H2Bub1 through RNF20 knockdown dramatically decreases H3K79 and H3K4 trimethylation in both normal and malignant lung epithelial cell lines. Concurrently, global transcriptional profiling analysis reveals that multiple tumor-associated genes such as CCND3, E2F1/2, HOXA1, Bcl2 modifying factor (BMF), Met, and Myc; and signaling pathways of cellular dedifferentiation, proliferation, adhesion, survival including p53, cadherin, Myc, and anti-apoptotic pathways are differentially expressed or significantly altered in these lung epithelial cells upon downregulation of H2Bub1. Moreover, RNF20 knockdown dramatically suppresses terminal squamous differentiation of cultured bronchial epithelial cells, and significantly enhances proliferation, migration, invasion, and cisplatin resistance of lung cancer cells. Furthermore, immunohistochemistry analysis shows that H2Bub1 is extremely low or undetectable in >70% of 170 lung adenocarcinoma samples. Notably, statistical analysis demonstrates that loss of H2Bub1 is significantly correlated with poor differentiation in lung adenocarcinoma (p = 0.0134). In addition, patients with H2Bub1-negative cancers had a trend towards shorter survival compared with patients with H2Bub1-positive cancers. Taken together, our findings suggest that loss of H2Bub1 may enhance malignancy and promote disease progression in lung adenocarcinoma probably through modulating multiple cancer signaling pathways.

Keywords: H2B monoubiquitination; RNF20; differentiation; global transcriptional profiling; lung adenocarcinoma; malignancy.

Figure 1

Downregulation of RNF20 mRNA in lung adenocarcinoma tissues and its association with histone modification in lung epithelial cell lines. (a) Box-and-Whisker plot of RNF20 mRNA level in lung adenocarcinoma (LUAD). Box represents first and third quartiles, thick band is median value, and bars extend to ± the interquartile range divided by the square root of the number of samples were applied to describe RNF20 gene expression values. Compared to normal lung tissues (Gr2, n=59), RNA20 mRNA was significantly decreased in LUAD (Gr1, n = 517) (fold change/FC = 0.86, and P= 9.12E-12). (b) Western blot analysis of RNF20, H2Bub1, H3K4/79-me3 and USP22 in lung epithelial cells at 72h post-transfection of control or RNF20 siRNA. Beta-actin served as the loading control.

Figure 2

Changes of gene expression profile and gene set in A549, H1299, H460 cells upon downregulation of H2Bub1. (a) Unsupervised hierarchical clustering analysis of global expression profiles in lung epithelial cells upon RNF20 knockdown. The two top rows represent two independent siRNA repeats, RNF is for RNF20 siRNA, and C is for control siRNA. (b) qRT–PCR and (c) Western blot analysis for selected genes. The level of each gene in cancer cell transfected with RNF20 siRNA is the average ratio of triplicate samples, and is presented as the ratio to control sample transfected with scramble siRNA (* P < 0.05, compared with control). (d). Selected gene sets that were enriched upon RNF20 knockdown in these lung cancer cells.

Figure 3

Knockdown of RNF20 enhances proliferation, migration and inhibits retinoic acid-induced squamous differentiation of BEAS-2B cells. (a) Enriched gene sets in BEAS-2B cells with silenced RNF20 and decreased H2Bub1 (P < 0.05, FDR < 5%). (b) Western blot for RNF20 and H2Bub1, p53, Myc, and ALDH1A1 proteins. (c) Enhanced cellular proliferation and (d) migration upon RNF20 knockdown in BEAS-2B cells. After 72 h of siRNA transfection, cell proliferation was measured. At 48 h post-transfection, 5 x 10⁴ cells transfected with either control or RNF20 siRNA were further subjected to transwell migration assay for 12 h, and the number of migrated cells was counted (*P < 0.05, compared to control siRNA). (e) qRT–PCR analysis of IVL, a squamous differentiation marker, mRNA in BEAS-2B cells transfected with control or RNF20 siRNA and cultured in the differentiating medium BEDM, data were presented as ratio to non-differentiating medium control (*P < 0.05, ** P < 0.01, compared with control). (f) Micrographs show morphology changes of BEAS-2B cells after transfection with control siRNA (left panel) or RNF20 siRNA (right panel) and then subcultured in BEDM. Arrows point to “fried egg” morphology of squamous cells in control siRNA-transfected BEAS-2B cells.

Figure 4

Impact of RNF20 knockdown on in vitro proliferation, migration, and invasion of lung cancer cell lines. (a) Western blot analysis of cell lysates of A549 and H460 cells transfected with either control or RNF20 siRNA and harvested at 72 h post-transfection. Actin served as loading control, total and phosphorylated Erk/Akt (T/P-Erk/Akt), p53, p21 E-Cadherin, and Vimentin were detected. The p53 blots were from separately developed blots for each cell, and the below Actin is its loading control. (b) Histogram of proliferation shows RNF20 knockdown increased the in vitro proliferation of both A549 and H460 cells over 72h (*P < 0.05, compared to control siRNA). (c) Representative photomicrographs of the migration chamber at the times indicated in control or RNF20 siRNA-transfected cells is shown on the left, with quantitative analysis of migrated cells shown on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, compared with control siRNA). (d) Lower surface of Matrigel transwell membranes seeded with cancer cells previously transfected with indicated siRNAs, 24 h after incubation (left), and corresponding quantitative analysis of invading cells (right). Data are shown as mean values graphed for indicated cells on the right (N = 3 replicates per cell type). Error bars show SD (*P < 0.05, ^**P < 0.01, compared with control siRNA).

Figure 5

Downregulation of H2Bub1 enhanced resistance to cisplatin in lung cancer cells. The levels of RNF20, H2Bub1, p21, p53, and apoptotic markers caspase-3/PARP cleaved products (T/C- Caspase-3/PARP for total and cleaved proteins) in (a) A549 and (b). H460 cells treated with cisplatin were analyzed by Western blot. Beta-actin was used as loading control. (c) Representative flow cytometry profile of A549 and H460 cells transfected with either control (left panel) or RNF20 siRNA (right panel). After cells were treated for 72 h with 20 uM Cisplatin, apoptosis was measured by flow cytometry analysis of Annexin-V (labeled with Alexa Fluor 488) staining in X axis and propidium iodide staining in Y axis. (d) Quantitative analysis of the experiments shows that apoptotic cells were significantly reduced in both A549 and H460 cells upon downregulation of H2Bub1. The experiment was repeated three times and data represent the average of the early apoptotic and late apoptotic cells (* P < 0.05; ** P < 0.01).

Figure 6

Loss of H2Bub1 and its association with cellular differentiation and survival of lung adenocarcinoma patients. Photomicrographs of H2Bub1 (upper panel) and total H2B (lower panel) in (a) normal lung tissue, and four representative lung adenocarcinoma tissues scored as (b) 0, (c) 1+, (d) 2+, (e) 3+ (Magnification × 100). (f). Kaplan–Meier analysis for overall survival of patients with H2Bub1 protein (Positive, n=91) or without H2Bub1 protein (Negative, n=79) (Neg v.s Pos, P = 0.237). (g). Kaplan–Meier analysis for recurrence free survival of lung adenocarcinoma patients with H2Bub1 protein (Positive, n=81) or without H2Bub1 protein (Negative, n=72) (Neg V.S Pos, P=0.103).