Fbxo4-mediated degradation of Fxr1 suppresses tumorigenesis in head and neck squamous cell carcinoma

Abstract

The Fbxo4 tumour suppressor is a component of an Skp1-Cul1-F-box E3 ligase for which two substrates are known. Here we show purification of SCF^Fbxo4 complexes results in the identification of fragile X protein family (FMRP, Fxr1 and Fxr2) as binding partners. Biochemical and functional analyses reveal that Fxr1 is a direct substrate of SCF^Fbxo4. Consistent with a substrate relationship, Fxr1 is overexpressed in Fbxo4 knockout cells, tissues and in human cancer cells, harbouring inactivating Fbxo4 mutations. Critically, in head and neck squamous cell carcinoma, Fxr1 overexpression correlates with reduced Fbxo4 levels in the absence of mutations or loss of mRNA, suggesting the potential for feedback regulation. Direct analysis reveals that Fbxo4 translation is attenuated by Fxr1, indicating the existence of a feedback loop that contributes to Fxr1 overexpression and the loss of Fbxo4. Ultimately, the consequence of Fxr1 overexpression is the bypass of senescence and neoplastic progression.

Fig. 1

Fbxo4 directly interacts with Fxr1. a Co-immunoprecipitation of Fbxo4 and Fxr1; arrows indicate Fbxo4 bands. b Endogenous Fbxo4 co-immunoprecipitates with Fxr1. c Ribbon diagram of the Fbxo4:Trf1 heterodimer, PDB:3L82. Fbxo4 is coloured in grey and Trf1 is in purple. d Intermolecular interactions between Fbxo4 and Trf1 derived from the PDB:3L82. e Ribbon diagram of the predicted interaction of Fbxo4 and an Fxr1 homology model. Fbxo4 is coloured in grey and Fxr1 is in purple. f Intermolecular interactions between Fbxo4 and Fxr1. HB is hydrogen bond, HYD is hydrophobic interaction, and ION an ionic bond. g Alignment of a semi-conserved motif in Trf1 and Fxr1. Identical amino acids are highlighted. Residues forming intermolecular bonds in Trf1 are boxed in blue, while residues mutated in this work are boxed in turquoise and magenta. Identity in this region was 30%, while similarity is 65%. h Fbxo4 E379A and E380A mutations disrupt the interaction between Fbxo4 and Fxr1. i Fbxo4 I377M mutation also disrupts the interaction between Fbxo4 and Fxr1. j Fxr1 V178A suppresses, while L189A Fxr1 enhances the interaction between Fbxo4 and Fxr1

Fig. 2

Fbxo4 ubiquitylates Fxr1 both in vivo and in vitro. a Fbxo4 reconstitution reduces Fxr1 levels in Fbxo4−/− MEFs. Note, arrow indicates Fbxo4; MEFs harbour a non-specific band that migrates below bona fide Fbxo4. b MLN-4924 rescues Fxr1 levels in both Fbxo4 + / + and −/− MEFs. c Fbxo4-induced Fxr1 ubiquitylation is enhanced by GSK3β co-expression and suppressed by MLN-4924 treatment for 6 h. d WT but not ΔF Fbxo4 ubiquitylates Fxr1 in vivo. e In vitro assay illustrates Fxr1 is ubiquitylated by Fbxo4. f Cyclin D1 ubiquitylation is used as a control for in vitro assays. g WT and S12E Fbxo4 enhance ubiquitylation of Fxr1 in vivo. h E379A, E380A and I377M Fbxo4 mutants lose the ability to ubiquitylate Fxr1 in vivo

Fig. 3

Genetic manipulation of Fbxo4 alters Fxr1 expression in HNSCC cells. a, b Oncomine analysis reveals elevated Fxr1 in human cancers (a) and a reverse correlation of Fbxo4 expression with Fxr1 in human HNSCC tissues and normal counterparts (b). All the data represent mean ± s.d. and were analysed by Student’s t test. Oncomine Box-and-Whisker plots: median values are shown as horizontal bars; the upper and lower part of the box show the 75th percentile and the 25th percentile, respectively; the upper and lower part of the bar show the 90th percentile and the 10th percentile, respectively; the points show outlier values. c Comparison of Fxr1 and Fbxo4 levels in OHKC and human HNSCC cells. Empty triangle indicates non-specific band observed in some human cell lines. d Quantification of Fbxo4 bands shown in c, normalised to β-Actin. e WT but not Fbxo4ΔF suppresses Fxr1 expression in SCC9 cells. The numbers below Fxr1 bands indicate the band quantification. f Fbxo4 knockdown triggers increased Fxr1 in HNSCC cells. Arrow indicates Fbxo4; empty triangle indicates nonspecific band. The numbers below Fxr1 bands indicate the band quantification. g Cycloheximide chase of Fxr1 levels in 74B cells following Fbxo4 knockdown. Arrow indicates Fbxo4; empty triangle indicates nonspecific band. h Quantification of Fxr1 turnover from g

Fig. 4

Fxr1 promotes cell proliferation and inhibits senescence-induced by ectopic Fbxo4 expression. a Fxr1 knockdown reverses Fbxo4 knockdown-induced cell proliferation of 74B cells. b Western blot shows Fbxo4 and/or Fxr1 Knockdown in 74B cells. Empty triangle indicates nonspecific band. c Expression of p21 and p27 in 74B cells upon Fbxo4 overexpression, Fxr1 knockdown and both Fbxo4 and Fxr1 overexpression. d β-Gal staining indicates senescent cells in 74B cells upon Fbxo4 overexpression, Fxr1 knockdown and both Fbxo4 and Fxr1 overexpression. The numbers show the percentage of β-Gal-positive cells in three independent experiments. e, f p21 and p27 knockdown rescue senescence in Fxr1 knockdown (e) and Fbxo4 overexpressing (f) 74B cells. The numbers show the percentage of β-Gal-positive cells in three independent experiments. g, i p21 and p27 knockdown rescues cell proliferation in Fxr1 knockdown (g) and Fbxo4 overexpressing (i) 74B cells. h, j Western blots show the knockdown of p21 and p27 in Fxr1 knockdown (h) and Fbxo4 overexpressing (j) 74B cells. Empty triangle indicates nonspecific band. All the data represent mean ± s.d. and were analysed by Two-way ANOVA, followed by Fisher’s LSD as post hoc test (n = 3). *p < 0.05; **p < 0.01. Scale bar, 10 μM

Fig. 5

Fxr1 expression reversely correlates with Fbxo4 protein levels in both human HNSCC and mouse papilloma tissues. a Representative image of Fbxo4 and Fxr1 IHC staining in human HNSCC TMAs. b Quantification and statistical analyses of the stained TMA specimens. Analysis was performed using nonparametric Mann–Whitney U test. **p < 0.01. c Representative illustration of Fxr1 IHC staining in papilloma-induced by NMBA in transgenic mice with Fbxo4 + / + , + /− and −/− genetic background. d, e Quantification and statistical analyses of the IHC stained normal (d) and papilloma (e) sections. Analyses were performed using nonparametric Kruskal–Wallis test. **p < 0.01. Box-and-Whisker plots: the upper and lower parts of the box show the 75th percentile and the 25th percentile, respectively; the bars outside of the box show the Min and Max values. Turquoise enclosed areas indicate normal epithelia; magenta-enclosed areas indicate either HNSCC or papilloma. Scale bar, 10 μM

Fig. 6

Negative feedback regulation of Fbxo4 by Fxr1. a Fbxo4 protein levels upon Fxr1 knockdown in 74A, 74B and SCC9 cells. b Fbxo4 mRNA levels upon Fxr1 knockdown in 74A, 74B and SCC9 cells. c Fbxo4 protein levels upon ectopic Fxr1 expression in U2OS and NIH3T3 cells. d Fbxo4 mRNA levels upon ectopic Fxr1 expression in HEK293T, U2OS and NIH3T3 cells. e Polysome profile in NIH3T3 cells with Fxr1 overexpression. f DNA gel shows the RT-PCR products from serial polysome fractions. g Quantitative distribution of Fbxo4 mRNA from (f). h Western blot from immunoprecipitation of Fxr1 in RIP analysis. i RIP analysis indicates Fxr1 interacts with Fbxo4 mRNA. TERC mRNA is used as positive control. All the data represent mean ± s.d. and were analysed by Student’s t test (n = 3). **p < 0.01

Fig. 7

Identification of the AREs that control Fbxo4 translation by Fxr1. a Schematic illustration of AREs in 3′-UTR of Fbxo4 mRNA; AREs are indicated in blue. ARE prediction is performed using AREsite2: http://rna.tbi.univie.ac.at/AREsite2/welcome. b Deletions made based on the ARE distribution. FL: full-length; Del: Deletion. c Fxr1 knockdown promotes the luciferase activity-mediated by both 3′-UTRs of p21 and Fbxo4 mRNAs in 74B cells. d Fxr1 overexpression suppresses the luciferase activity-mediated by 3′-UTR of Fbxo4 mRNA in HEK293T cells. e ARE deletion rescues luciferase activity in HEK293T cells with Fxr1 overexpression. f ARE deletion reduces luciferase activity in 74B cells with Fxr1 knockdown. g The comparison of basal luciferase activity with full-length and deleting 3′-UTRs of Fbxo4 mRNA in 74B cells. h Proposed model summarises the regulation of Fxr1 by Fbxo4 and feedback regulation of Fbxo4 by Fxr1. All the data represent mean ± s.d. c, d were analysed by Student’s t test (n = 3). e–g were analysed by One-way ANOVA, followed by Fisher’s LSD as post hoc test (n = 3). **p < 0.01