Epigenetic inactivation of the p53-induced long noncoding RNA TP53 target 1 in human cancer

Abstract

Long noncoding RNAs (lncRNAs) are important regulators of cellular homeostasis. However, their contribution to the cancer phenotype still needs to be established. Herein, we have identified a p53-induced lncRNA, TP53TG1, that undergoes cancer-specific promoter hypermethylation-associated silencing. In vitro and in vivo assays identify a tumor-suppressor activity for TP53TG1 and a role in the p53 response to DNA damage. Importantly, we show that TP53TG1 binds to the multifaceted DNA/RNA binding protein YBX1 to prevent its nuclear localization and thus the YBX1-mediated activation of oncogenes. TP53TG1 epigenetic inactivation in cancer cells releases the transcriptional repression of YBX1-targeted growth-promoting genes and creates a chemoresistant tumor. TP53TG1 hypermethylation in primary tumors is shown to be associated with poor outcome. The epigenetic loss of TP53TG1 therefore represents an altered event in an lncRNA that is linked to classical tumoral pathways, such as p53 signaling, but is also connected to regulatory networks of the cancer cell.

Keywords: DNA methylation; cancer; epigenetics; long noncoding RNA.

Fig. 1.

Epigenetic silencing of the lncRNA TP53TG1 in cancer cells. (A) Schematic strategy used to identify tumor-specific DNA methylation events in lncRNAs. (B) Bisulfite genomic sequencing analysis of TP53TG1 promoter CpG island in cancer cell lines and normal tissues. Locations of bisulfite genomic sequencing PCR primers (black arrows), CpG dinucleotides (vertical lines), and the TSS (long black arrow) are shown. Ten single clones are represented for each sample. The presence of unmethylated and methylated CpGs is indicated by white and black squares, respectively. Red circles indicate the CpGs detected by the DNA methylation 450K microarray. (C) DNA methylation-associated transcriptional silencing of TP53TG1 in cancer cells. (Upper) TP53TG1 expression levels in methylated (HCT-116, KM12, and KATOIII) and unmethylated (SW480, HT29, MKN-7, SNU-1, MKN-45, NUGC-3, and GCIY) cancer cell lines determined by qRT-PCR. (Lower) Restored TP53TG1 expression after treatment with the DNA demethylating agent 5-aza-2´-deoxycytidine (AZA) or upon genetic depletion (DKO) in the originally methylated cell lines. Values were determined from triplicates and are expressed as the mean ± SEM. (D) TP53TG1 RNA-FISH and intracellular localization. (Upper) TP53TG1 subcellular distribution in DKO by qRT-PCR. RNAU6b and GAPDH genes were used as controls for the nuclear and cytoplasmic fractions, respectively. Values were determined from triplicates and are expressed as the mean ± SEM. The effectiveness of cell fractionation was evaluated with lamin B1 (nuclear) and tubulin (cytoplasmic) by Western blot. C, cytoplasm; N, nucleus. (Lower) Single-molecule visualization of TP53TG1 (red spots) in HCT-116 and DKO cell lines by FISH.

Fig. 2.

TP53TG1 exhibits tumor-suppressor features in vitro and in vivo. (A) Efficient restoration of TP53TG1 upon transfection in HCT-116 cells according to qRT-PCR. Expression of TP53TG1 in cellular pools (four different clones) of HCT-116 stable transfected cells in comparison with the empty vector. Values were analyzed from triplicates and expressed as the mean ± SEM. (B) MTT and colony formation assays. TP53TG1 transfection reduces cell viability and the number of colonies in HCT-116 cells. (C) TP53TG1 restoration induces apoptosis in HCT-116 cells. Apoptotic cells were evaluated in TP53TG1 stably transfected HCT-116 cells after 24 h as the sub-G1 cell population and annexin V-positive/IP-negative cells by FACS analysis. Caspase 3/7 activity was determined after 24 h by a luminometric assay. Values are expressed as the mean ± SEM (n = 6). (D) TP53TG1 reduces invasion and migration properties in HCT-116 cells evaluated by the xCELLigence Real-Time (cell index) approach (Left and Middle) and the wound-healing assays (n = 3) (Right), respectively. (E) Growth inhibitory effect of TP53TG1 restitution in HCT-116 mouse tumor xenografts. (Left) Tumor volume (n = 8) was monitored over time. (Middle Left) Tumors were excised and weighed at 34 d. (Middle) Representative images of the confluence of HCT-116 stable transfected cells maintained in vitro for 7 d after implantation in mice and the size of the tumors at the end of the analyses. (Middle Right) Detection and (Right) quantification of apoptotic cells (in brown) from s.c. tumors by TUNEL assay (n = 5). (Scale bar, 100 μm.) (F) Growth-inhibitory effect of TP53TG1 restitution in HCT-116 cells in a colorectal orthotopic mouse model. Small pieces of the s.c. tumor model were implanted in the colon of nude mice, and tumor weight was measured after 30 d (n = 4–5). (G) Effect of TP53TG1 RNAi-mediated knockdown in the nontumorigenic HCEC line. (Left) Values were obtained by qRT-PCR and expressed as the mean ± SEM (n = 3). The photograph shows the transfection control efficiency (green). TP53TG1 down-regulation after 72 h reduces cell viability and apoptosis. Cell viability (Middle Left) was determined by MTT (n = 3) and the frequency of apoptotic cells was determined by FACS analysis of the sub-G1 cell population (n = 3) (Middle Right) and annexin V-positive/IP-negative cells (Right) (n = 2 for each independent siRNA). **P < 0.01; *P < 0.05.

Fig. 3.

DNA damage induces TP53TG1 expression in a p53-dependent manner. (A) Treatment with DNA-damaging agents increases p53 and TP53TG1 expression in TP53TG1-unmethylated and p53 wild-type HCECs. The cells were treated with doxorubicin (100 nM) or etoposide (50 µM) for 24 h. Values were obtained by qRT-PCR and expressed as the mean ± SEM (n = 3). (B) Treatment with DNA-damaging agents increases TP53TG1 expression in the TP53TG1-unmethylated and p53 wild-type colorectal (SW-48) and gastric (SNU-1) cancer cells but not in TP53TG1-unmethylated and p53 mutant colon cancer cells (SW-620). Cells were treated with bleomycin, cisplatin, and etoposide for 24 h. Expression was analyzed in triplicates by qRT-PCR, and the results are expressed as the mean ± SEM (n = 4). (C) TP53TG1 induction upon DNA damage depends on wild-type p53. p53 silencing by siRNA in HCECs prevents TP53TG1 activation upon 50 µM etoposide treatment for 24 h. Values were obtained by qRT-PCR and expressed as the mean ± SEM (n = 3). (D) The increase of TP53TG1 after DNA damage is mediated by direct binding of the p53 protein to p53 response elements (p53 REs) of the TP53TG1 gene. After doxorubicin (100 nM) treatment for 24 h in HCECs, ChiP was performed with IgG or p53 antibodies, followed by qPCR and semiquantitative PCR in the p53 RE region. Values of qPCR were obtained from triplicates and expressed as the mean ± SEM (n = 3). (E) Recovery of TP53TG1 expression restores chemosensitivity to DNA-damaging agents. TP53TG1 stably transfected HCT-116 cells were treated with various DNA-damaging anticancer drugs, and cell viability was determined by MTT. The half-maximal inhibitory concentration (IC₅₀) was calculated and expressed as relative units. (F) Orthotopic tumors derived from TP53TG1-transfected HCT-116 cells were more sensitive to 5-fluorouracil + oxaliplatin treatment than empty vector-derived tumors according to tumor weight. Significance of Mann-Whitney U test, ***P < 0.001; **P < 0.01; *P < 0.05.

Fig. 4.

TP53TG1 binds to the YBX1 protein. (A) Detection of candidate TP53TG1-associated proteins by RNA pull-down assays. In vitro-synthesized biotinylated full-length TP53TG1 lncRNA and other RNA control sequences were incubated in the presence of total HCEC extracts, retrieved with streptavidin beads, and the associated proteins were analyzed by SDS/PAGE. A specific band of ∼45 KDa (black square) was pulled down by TP53TG1 lncRNA. TP53TG1 antisense and unrelated RNA (Uc.160) were used as negative controls. (B) Identification of the TP53TG1 RNA isolated pull-down band by MS. The specific band detected in the RNA pull-down assay was cut out and trypsin-digested for MS analysis. The spectra show the four YBX1 peptides that bind to TP53TG1 lncRNA. (C) Western blot showing the specific association between YBX1 and TP53TG1 lncRNA in the samples obtained from the RNA pull-down. TP53TG1 antisense RNA and an unrelated RNA (Uc.160) were used as negative controls. Total extract of the TP53TG1 unmethylated HCEC line was used as the input control. (D) Confirmation of the YBX1 interaction with TP53TG1 by YBX1 immunoprecipitation (reverse pull-down). Endogenous YBX1 was immunoprecipitated from total HCEC extracts with anti-YBX1 antibody. The pulled-down RNA was extracted and analyzed by (Left) RT-PCR and (Right) qRT-PCR. The G3BP1 RNA was used as a positive target of YBX1. (E) YBX1 binds to the central region of TP53TG1. (Upper and Lower Left) Various truncated RNA fragments of TP53TG1 were pulled down following incubation with HCEC total extract, and YBX1 protein was detected by Western blot. TP53TG1 antisense and an unrelated RNA were used as negative controls. (Upper Right) Increasing amounts of recombinant YBX1 protein were incubated with either the 3′ end or the central region of the TP53TG1 transcript and run on a native 6% (wt/vol) acrylamide gel. The mobility shift indicates the direct interaction between YBX1 and TP53TG1. (Lower Middle) The middle sequence of TP53TG1 with the five YBX1 binding sites that were mutated is indicated. (Lower Right) RNA pull-down experiments show that mutation of these sites impairs the interaction. (F) Transfection of the TP53TG1 mutant form (Upper Left), unable to bind to the YBX1 protein, in HCT-116 cells does not affect growth determined by (Upper Right) MTT and (Lower) colony formation assays. (G) Cotransfection of the full-length wild-type YBX1 protein in TP53TG1-transfected HCT-116 cells increases (Upper) their growth (MTT) and (Lower) invasiveness (xCELLigence Real-Time). Values were expressed as the mean ± SEM (n = 3). ***P < 0.001; *P < 0.05.

Fig. 5.

TP53TG1 binds to the YBX1 protein, preventing its nuclear localization. (A) Lack of association between TP53TG1 and YBX1 expression levels. YBX1 expression levels were analyzed by qRT-PCR and Western blot after TP53TG1 stable overexpression in HCT-116 (four replicates: 1–4) and after TP53TG1 silencing by RNAi in HCECs. Expression levels of the TP53TG1 and YBX1 genes were determined by qRT-PCR in human colon and gastric cancer cell lines. Values of qRT-PCR were obtained from triplicates and expressed as the mean ± SEM (n = 3). (B) Recovery of TP53TG1 expression by transfection in the methylated HCT-116 cells modifies the subcellular distribution of the YBX1 protein. TP53TG1 restoration excludes the YBX1 protein from the nucleus according to subcellular fractionation experiments followed by Western blot (Left) and immunofluorescence assays (Right). (Scale bar, 5 µm.) White triangles indicate representative YBX1 expression in the nucleus. (C) Immunofluorescence assays confirm the nuclear exclusion of the YBX1 protein in the TP53TG1 unmethylated colorectal (HT-29, SW480, and HCEC) and gastric (GCIY and NUG-3) cell lines. Conversely, TP53TG1 hypermethylated and silenced colorectal (KM12) and gastric (TGBC11TKB and KATO-III) cell lines show nuclear localization, in addition to the cytosolic staining. (D) Quantification of nuclear immunofluorescence intensity (mean gray value) in the TP53TG1-unmethylated (HT-29, SW480, HCEC, GCIY, and NUG-3) and -methylated (HCT-116, KM12, TGBC11TKB, and KATO-III) cell lines. In the box plot, the central line represents the median and the limits show the upper and lower percentiles. Mann–Whitney U test, *P = 0.0317. M, methylated; U, methylated.

Fig. 6.

TP53TG1 re-expression damps the YBX1-activated PI3K/AKT pathway and promotes sensitivity to targeted small drugs. (A) ChIP for the YBX1 protein in the PI3K promoter region. Restoration of TP53TG1 expression in HCT-116 cells inhibits the binding of the YBX1 protein to the PI3K regulatory region. (B) Western blot showing (Left) reduced expression of PI3K and lower phosphorylation levels of its downstream target AKT upon restitution of TP53TG1 expression in HCT-116 cells. (Right) Transfection of the TP53TG1 mutant form does not change PI3K expression or AKT phosphorylation levels. (C) Recovery of TP53TG1 expression enhances the sensitivity to (Left) PI3K and (Right) AKT small drug inhibitors. TP53TG1 or empty vector-transfected HCT-116 cells were treated for 48 h with KU-55933 (PI3K inhibitor) or perifosine (AKT inhibitor). Cell viability was measured by the MTT assay. Values of the half-maximal inhibitory concentration (IC₅₀) are shown. (D) Western blot showing (Left) reduced phosphorylation of MDM2 and stabilization of its target p53 upon expression of TP53TG1 in HCT-116 cells. (Right) Transfection of the TP53TG1 mutant form does not induce these changes. ***P < 0.001; **P < 0.01.

Fig. 7.

Occurrence and impact of TP53TG1 hypermethylation in gastrointestinal cancer patients. (A) Frequency of TP53TG1 hypermethylation in primary colorectal and gastric tumors derived from TCGA and other publicly available datasets. (B) The presence of TP53TG1 methylation is significantly associated with loss of expression of the TP53TG1 transcript in primary gastric tumors from TCGA. The box plots illustrate the distribution of RNA-seq expression values; the central solid line indicates the median; the limits of the box show the upper and lower percentiles. Mann–Whitney U test, ***P < 0.0001. (C) Kaplan–Meier curves showing that the presence of TP53TG1 hypermethylation in gastric cancer patients (n = 63) is significantly associated with shorter PFS (P = 0.018). CI, confidence interval; HR, hazard ratio; M, methylated TP53TG1; U, unmethylated TP53TG1. (D) TP53TG1 hypermethylation is an independent prognostic factor of shorter PFS in gastric patients with locoregional disease (stages I and II). Kaplan–Meier curves show that the presence of TP53TG1 hypermethylation in this group (n = 43) is significantly associated with shorter PFS (P = 0.028). (E) Forest plot representation of the Cox proportional hazard regression models, showing that TP53TG1 methylation is an independent prognostic factor of worse PFS (HR = 3.64, 95% CI = 1.19–11.11, P = 0.023) than other parameters such as gender, age, histology, and location.