β-Trcp ubiquitin ligase and RSK2 kinase-mediated degradation of FOXN2 promotes tumorigenesis and radioresistance in lung cancer

Abstract

Aberrant expression of FOXN2, a member of the Forkhead box transcription factors, has been found in several types of cancer. However, the underlying mechanisms of FOXN2 deregulation in tumorigenesis remain largely unknown. Here, we find that FOXN2 binds to and is ubiquitinated by β-Trcp ubiquitin ligase and RSK2 kinase for degradation. Furthermore, we demonstrate that the Ser365 and Ser369 sites in a conserved DSGYAS motif are critical for the degradation of FOXN2 by β-Trcp and RSK2. Moreover, gain-of-function and loss-of-function studies show that FOXN2 impairs cell proliferation in vitro and in vivo and enhances the radiosensitivity of lung cancer. Importantly, β-Trcp-mediated and RSK2-mediated degradation of FOXN2 promotes tumorigenesis and radioresistance in lung cancer cells. Collectively, our study reveals a novel post-translational modification of FOXN2 and suggests that FOXN2 may be a potential therapeutic and radiosensitization target for lung cancer.

Fig. 1

FOXN2 interacts with β-Trcp in cells. a Immunoblot analysis of whole cell lysates (WCL) and immunoprecipitates (IP) lysed from HEK293T cells transfected with plasmids as indicated. b H1299 cells were transfected with indicated constructs. Co-IP experiments were performed using S-protein beads and blotted with indicated antibodies. c The endogenous interaction between β-Trcp and FOXN2 was detected by Co-IP. Rabbit IgG was used as a negative control

Fig. 2

FOXN2 stability is negatively regulated by β-Trcp. a β-Trcp silencing leads to the accumulation of endogenous FOXN2. Cells were transfected with indicated siRNAs, and lysates were subjected to western blotting with the indicated antibodies. b Western blotting analysis of WCL derived from H1299 cells with or without MG132 treatment. c Left panel: H1299 cells were transfected with the indicated plasmids and harvested at the indicated points after CHX with or without MG132 treatment for immunoblotting. Right panel: quantification of the FOXN2 band intensity is shown. d Left panel: H1299 cells were transfected with the indicated siRNAs. At 48 h post transfection, cells were treated with 20 μg/mL of cycloheximide (CHX) and harvested at the indicated time points. Western blotting was conducted to detect the protein level of FOXN2. Right panel: quantification of the FOXN2 band intensity is shown

Fig. 3

The Ser365 and Ser369 sites in a conserved DSGYAS motif are responsible for the ubiquitination of FOXN2 by β-Trcp. a Alignment of the candidate phosphodegron sequence in FOXN2 in different species. b HEK293T cells were transfected with the indicated plasmids and harvested 24 h after transfection. Co-IP experiments were performed using S-protein beads and blotted with the indicated antibodies. c HeLa cells transfected with plasmids as indicated. At 24 h post transfection, cells were treated with MG132 (10 μM) for 4 h before harvesting. The lysates were incubated with S beads and then subjected to immunoblot. d HeLa cells were transfected with Myc-FOXN2-WT or Myc-FOXN2 AA plasmid. At 24 h post transfection, cells were treated with 20 μg/mL of cycloheximide (CHX) and collected at the indicated time points. Western blotting was performed to examine the protein level of FOXN2. e Quantification of the FOXN2-WT and FOXN2-AA band intensity in d

Fig. 4

RSK2 binds to FOXN2 and promotes its ubiquitination and degradation. a H1299 cells were transfected with the indicated constructs, followed by IP with S-protein beads and immunoblotting with the indicated antibodies. b H1299 cells were transfected with SFB-tagged RSK2 plasmid. After 24 h, cells were collected and then incubated with S-protein beads. The samples were analyzed by western blotting with the indicated antibodies. c H1299 cells were harvested and subjected to immunoprecipitation using anti-IgG or anti-RSK2 antibody and then analyzed by western blotting. d Immunoblot analysis of H1299 and A549 cells transfected with the indicated siRNAs. e RSK2 depletion inhibits FOXN2 ubiquitination in vivo. HeLa cells were transfected with the indicated siRNAs and plasmids, followed by the treatment with MG132 (10 μM) for 4 h prior to collection. The lysates were incubated with S beads overnight and then subjected to immunoblotting to examine the polyubiquitylation of exogenous FOXN2. f Upper panel: H1299 cells were transfected with the indicated siRNAs. At 48 h post transfection, cells were treated with 20 μg/mL of cycloheximide (CHX) and harvested at the indicated time points. Western blotting was conducted to detect the protein level of FOXN2. Lower panel: quantification of the FOXN2 band intensity is shown

Fig. 5

FOXN2 knockdown results in cell cycle redistribution in two lung cancer cells. a Gene set enrichment analysis (GSEA) of the primary lung cancer database from the Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/). The enriched pathway analysis shows that FOXN2 is associated with ubiquitin-mediated proteolysis, cell cycle, mismatch repair, nucleotide excision repair, and homologous recombination. b Western blotting shows the knockdown efficiency of FOXN2 by two different siRNAs in H1299 and A549 cells. c Cell cycle analysis of H1299 and A549 cells after transfection with the indicated siRNAs for 48 h (left panel). Histograms show the percentage of cells in different phases (right panel). **P < 0.01 compared to control cells

Fig. 6

Loss of FOXN2 promotes lung cancer cell growth in vitro and in vivo. a FOXN2 depletion promoted cell growth. Cells were transfected with the indicated siRNAs for 48 h and then seeded at low density in plates. The cell numbers were counted every other day. b Colony formation was significantly increased in FOXN2-depleted cells. Cells transfected with the indicated siRNAs were grown for 2 weeks. Representative violet staining pictures are shown (upper panel). The numbers of colonies were counted and shown with histograms (lower panel). ***P < 0.001 compared to control cells. c Western blotting shows the knockdown efficiency of FOXN2 by two different shRNAs in H1299 cells. d FOXN2 inhibition promotes tumor growth in vivo. Representative images of the xenograft tumors in three groups are shown. e The growth curves of tumors in the three groups are presented (five mice/group). f Tumor weights in the three groups are shown (five mice/group)

Fig. 7

FOXN2 enhances the radiosensitivity of lung cancer cells. a FOXN2-depleted H1299 cells (upper panel) and A549 cells (lower panel) were irradiated with 2 Gy and collected at the indicated time points. An immunostaining experiment was performed to examine γ-H2AX foci formation. DAPI was applied to counterstain the nuclei. b Quantification of the γ-H2AX foci result is shown in a. More than 100 cells were counted in each group, and data are presented as means ± SD. ***P < 0.001 compared to control cells. c Radioresistance of cells lacking FOXN2. H1299 cells (left panel) and A549 cells (right panel) were transfected with control or FOXN2 siRNAs and irradiated with doses as indicated. The colonies were counted 14 days later. Experiments were performed in triplicate, and the results are presented as the means ± SD

Fig. 8

β-Trcp and RSK2-mediated degradation of FOXN2 promotes cell proliferation and radioresistance in lung cancer cells. a FOXN2 knockdown cells were reconstituted with either FOXN2-WT (wild type) or FOXN2-AA mutant as indicated. b The growth curve of H1299 cells is shown. Cells were transfected with the indicated siRNAs and plasmids and then seeded at low density in plates. The cell numbers were calculated every other day. c H1299 cells were transfected with the indicated siRNAs, and plasmids were allowed to grow 2 weeks to form colonies. Representative violet staining results are shown (upper panel). The numbers of colonies were counted and shown with histograms (lower panel); **P < 0.01. d H1299 cells transfected with the indicated siRNAs and plasmids were irradiated with 2 Gy and harvested at the indicated time points. An immunostaining experiment was carried out to detect the γ-H2AX foci formation. e Quantification of γ-H2AX foci is shown in d; **P < 0.01, ***P < 0.001. f Cell survival curves derived from H1299 cells. Cells were transfected with the indicated siRNAs and plasmids and treated with the radiation dose as indicated. Percentages of surviving colonies were measured 2 weeks later. The experiments were conducted in triplicate, and the results are presented as the means ± SD