RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC

Abstract

RNA-binding proteins (RBP) regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1), plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4) RNA structure within) both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1) and the non-coding RNA Telomerase RNA Component (TERC), and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells.

Fig 1. FXR1 is overexpressed in head and neck squamous cell carcinoma.

(A) Comparison of altered copy number and mRNA expression level of RBPs in TCGA HNSCC data sets. (B) Percent mRNA expression level of top altered RBPs in 279 TCGA HNSCC tumor samples. Frequencies of expression levels are shown as a percentage of all cases. (C) Representative dual color FISH of FXR1 gene (green spots) and a chromosome 3 loci probe (red spots) in HNSCC (scale bar 5μm). (D) FXR family of proteins (FXR1, FXR2 and FMR1) coding mRNA expression levels in TCGA database where cancer genome browser was used to visualize the data sets. N = Normal; and T = Tumor. (E) qRT-PCR estimate of mRNA levels of FXR proteins in eight matched HNSCC tumor compared to normal adjacent tissue samples. (F) Immunoblot analysis of eight HNSCC and matched adjacent normal tissue for FXR1, FXR2, FMR1, and GAPDH. (G) qRT-PCR analysis of mRNA levels of FXR1, FXR2 and FMR1 in eight HNSCC cell lines compared to HOK. (H) Immunoblot analysis of FXR1, FXR2, and FMR1 in HNSCC cell lines compared to HOK. GAPDH serves as a loading control.

Fig 2. Silencing of FXR1 in cancer cells promotes cellular senescence.

(A) After 72hrs of shRNA treatment, UMSCC74A and 74B FXR1 KD and control cells are fixed and stained with propidium Iodide (PI), and analyzed by fluorescence-activated cell sorting to evaluate the number of cells in different stages of cell cycles. (B) 72 hours post-transduction staining for SA-β-gal in FXR1 depleted UMSCC74A and 74B cells. Three independent fields for each experiment are used here for quantification. (C) Measurement of MUG conversion to 4-MU by senescence associated β-galactosidase in FXR1 depleted UMSCC74A and 74B cells. (D) Representative immunofluorescence images of pATM and γ-H2AX staining FXR1 depleted UMSCC74A and 74B cells compared to control. Quantification of foci containing cells is given under each cell lines. (*p<0.05, **p<0.005, ***p<0.0005).

Fig 3. Depletion of FXR1 alters cell cycle proteins in HNSCC.

(A) Relative quantity of RNAs extracted from control and FXR1 KD cells are estimated by using qRT-PCR. GAPDH serves as a control. (B) Immunoblot analysis of senescence marker proteins in both FXR1 depleted UMSCC74 and 74B cells. β-Actin used as a loading control. (C) Relative quantity of p21 and p53 mRNAs, extracted from shcontrol and shFXR1 treated P53wt and P53mut cells, are estimated by using qRT-PCR. GAPDH serves as a control. (D) Western blot analysis of FXR1 depleted p53Wt and mutant oral cancer cells. GAPDH is used as a loading control. (E) FXR1 depleted p53Wt and mutant oral cancer cells stained with SA-β-gal. (F) Western blot yH2AX expression in UMSCC74A and UMSCC74B cells upon FXR1 KD. (G) Protein lysates of UMSCC74B cells are subjected to RNP IP followed by qRT-PCR analysis to measure the relative quantities of RNAs in FXR1 IP compared with control IgG IP. GAPDH serves as a loading control. (H) RT-PCR of products acquired from inputs and IPs respectively from Mouse IgG as control (Lanes 1, 3) and IP of FXR1 (Lanes 2, 4) were separated and visualized by agarose gel electrophoresis. It clearly shows that p21 mRNA and TERC RNA are enriched in the IP samples, whereas p27 is not. NEB 50bp DNA ladder #N3236S (L) was loaded as a molecular marker. (*p<0.05, **p<0.005, ***p<0.0005).

Fig 4. FXR1 destabilizes p21 mRNA.

(A) FISH analysis of p21 in a HNSCC TMA. Green indicates p21 and red denotes the control loci (scale bar 5μm). (B) Relative p21 expression data obtained from cancer genome browser (N-43, T-521) (S4 Table). (C) Relative mRNA quantity of p21 in eight matched HNSCC tumor compared to normal adjacent tissue samples estimated using qRT-PCR. GAPDH serves as a control. (D) Immunoblot analysis of p21 protein from eight representative matched HNSCC tumor and normal adjacent samples. GAPDH is used as a loading control. (E) Relative quantity of FXR1 and p21 levels estimated by qRT-PCR in UMSCC74B cells after treatment with FXR1 shRNA. Cells were collected at indicated time points. FXR1 and p21 levels in shControl treated UMSCC74B cells were taken as 1 for each time points. (F) Immunoblot analysis of p21 protein at different time points, as, mentioned in Fig 4E. (G) The mRNA decay rate of p21 as indicated in UMSCC74B cells by qRT-PCR after silencing FXR1 followed by transcription inhibition with actinomycin-D for mentioned time points in the graph. Actin serves as a control. Data here represents the mean of n  =  3 experiments. (H) Forty-eight hours after transfection of UM74B FXR1 KD and control cells with empty 3’UTR luciferase plasmid, luciferase-fused GAPDH 3′UTR plasmid or different segments of P21 3′UTR, the lysates were analyzed for luciferase activity using luminometer. The empty 3’UTR luciferase plasmid and luciferase-fused GAPDH 3′UTR served as a transfection and loading control. Values are the means ± SD from three independent experiments by using unpaired two sample t-test. (I) Binding of FXR1 with the 3′UTR of p21seg1 and p21seg2 RNAs at the G4 region. RNP IP was performed 48 h post-transfection of UMSCC74B cells with seg1 and seg2 3′UTR fused to a luciferase reporter construct. Luciferase mRNA was detected using qRT-PCR. The luciferase gene in the empty-3′UTR was used as a transfection and qRT-PCR control. (*p<0.05, **p<0.005, ***p<0.0005).

Fig 5. FXR1 stabilizes TERC RNA.

(A) FISH analysis of TERC DNA in a HNSCC TMA. Green indicates TERC DNA and red denotes control loci (scale bar 5μm). (B) Relative expression of TERC estimated by qRT-PCR in eight matched HNSCC and normal adjacent tissue samples. (C) Relative expression of FXR1 and TERC levels are simultaneously estimated in UMSCC74B cells under treatment of FXR1 shRNA for indicated time points. FXR1 and TERC levels in shControl treated UMSCC74B cells were taken as 100% for each time points. (D) The RNA decay rate of TERC as indicated in UMSCC74B cells by qRT-PCR after silencing FXR1 followed by transcription inhibition with actinomycin-D for mentioned time points in the graph. Actin serves as a control. Data here represents the mean of n  =  3 experiments. (E) Forty-eight hours after transfection of UM74B FXR1 KD and control cells with empty 3’UTR luciferase plasmid, luciferase-fused GAPDH 3′UTR plasmid or two segments of TERC, the lysates were analyzed for luciferase activity using luminometer. The empty 3’UTR luciferase plasmid and luciferase-fused GAPDH 3′UTR served as a transfection and loading control. Values are the means ± SD from three independent experiments by using unpaired two sample t-test. (F) Association of FXR1 with the TERC and TERCmut RNAs. RNP IP was performed 48 h post-transfection of UMSCC74B cells with TERC and TERCmut fused to a luciferase reporter construct. Luciferase mRNA was detected using qRT-PCR. The luciferase gene in the empty-3′UTR was used as a transfection and qRT-PCR control. (G) TRAP assay is done on protein extracts (0.05μg) from FXR1 KD and control UMSCC74B cells. Heat inactivated controls; negative, positive, and loading controls are also shown in the figure. (H) Quantification of internal controls and telomerase activity bands (*) from FXR1 KD and control UMSCC74B cells. The values were normalized against the internal control bands from the heat inactivated samples from the above mentioned samples. P values are calculated from 4 independent experiments. (*p<0.05, **p<0.005, ***p<0.0005).

Fig 6. FXR1 involves both p21 and TERC RNA to promote senescence.

(A) qRT-PCR analyses of RNA levels of UMSCC74B cells transfected with single or combination of p21 overexpression plasmid or siTERC RNA. GAPDH serves as a loading control. (B) Immunoblot of protein expression levels of FXR1 and p21 in UMSCC74B cells transfected independently or together with p21 overexpression plasmid or siTERC. GAPDH serves as a loading control. (C) Quantification of the western blot shown in Fig 6B. (D) Staining of SA-β-gal in UMSCC74B cells transfected independently or together with p21 overexpression plasmid or siTERC. (E) Quantitative values of MUG conversion to 4-MU by senescence associated β-galactosidase for Fig 6D. (F) SA-β-gal staining of shControl and shFXR1 treated UMSCC74B cells, also co-transduced and/or co-transfected together or independently with shp21 or TERC overexpression plasmid. (G) qRT-PCR analyses of TERC RNA levels in shControl and shFXR1 treated UMSCC74B cells also co-transduced and/or co-transfected together or independently with shp21 or TERC overexpression plasmid. GAPDH serves as a loading control (n = 2). Statistical significance for TERC overexpressing cells from a plasmid-borne copy is not calculated. (H) Immunoblot of protein expression levels of FXR1 and p21 in shControl and shFXR1 treated UMSCC74B cells also co-transduced and/or co-transfected together or independently with shp21 or TERC overexpression plasmid. β-Actin serves as a loading control. (*p<0.05, **p<0.005, ***p<0.0005).

Fig 7. FXR1-regulated senescence is irreversible.

(A) Relative quantity of FXR1, p21, and TERC are quantified by qRT-PCR under non-inducible, Inducible and reverse inducible FXR1 silencing conditions in UMSCC74B cells. (B) Immunoblot analysis of protein levels of UMSCC74B cells as described above. (C) The UMSCC74B cells are subjected to SA-β-gal staining as described in Fig 7A. (D) Representative culture dishes from clonogenic assays of cells transfected with indicated conditions. (E) The panel depicts the colony forming efficiency from clonogenic assays of UMSCC74B cells. The data are presented as the means ± S.D. from three independent experiments. (F) Model representation of evasion of cellular senescence through FXR1 by destabilizing p21 and stabilizing TERC in conjunction with activation of p53. (*p<0.05, **p<0.005, ***p<0.0005).