Long Noncoding RNA PURPL Suppresses Basal p53 Levels and Promotes Tumorigenicity in Colorectal Cancer

Abstract

Basal p53 levels are tightly suppressed under normal conditions. Disrupting this regulation results in elevated p53 levels to induce cell cycle arrest, apoptosis, and tumor suppression. Here, we report the suppression of basal p53 levels by a nuclear, p53-regulated long noncoding RNA that we termed PURPL (p53 upregulated regulator of p53 levels). Targeted depletion of PURPL in colorectal cancer cells results in elevated basal p53 levels and induces growth defects in cell culture and in mouse xenografts. PURPL associates with MYBBP1A, a protein that binds to and stabilizes p53, and inhibits the formation of the p53-MYBBP1A complex. In the absence of PURPL, MYBBP1A interacts with and stabilizes p53. Silencing MYBBP1A significantly rescues basal p53 levels and proliferation in PURPL-deficient cells, suggesting that MYBBP1A mediates the effect of PURPL in regulating p53. These results reveal a p53-PURPL auto-regulatory feedback loop and demonstrate a role for PURPL in maintaining basal p53 levels.

Keywords: CRC; HuR; LINC01021; LOC643401; MYBBP1A; PURPL; RP11-46C20.1; lincRNA; lncRNA; p53.

Figure 1. RNA-seq from multiple CRC lines identifies PURPL as a p53-regulated lncRNA

RNA-seq was performed from isogenic (p53WT and p53KO) HCT116, RKO and SW48 cells. (A) Scatter plot (left), Rank order of gene expression (right) and RNA-seq snapshot. (B) p53-dependent induction of PURPL (RP11-46C20.1) after DOXO treatment. (C) RT-qPCR for PURPL from nuclear and cytoplasmic fractions of untreated HCT116 cells. The cytoplasmic GAPDH mRNA and nuclear lncRNA MALAT1 were used as controls.

Figure 2. Targeted disruption of the p53RE in PURPL uncovers a pro-survival function of PURPL

(A) (Top) Schematic showing the p53RE in the PURPL promoter and location of the guide RNAs. (Bottom) RT-qPCR analysis from HCT116 PURPL-WT cells (WT#1 and WT#2) and HCT116 PURPL-KO cells (KO#1 and KO#2) untreated or treated with DOXO for 16 hr. (B) PURPL-WT and PURPL-KO cells were untreated or treated with DOXO for 48 hr; cell death (sub-G1 cells) and effect on cell cycle was assessed by PI staining followed by FACS analysis. (C) Immunostaining for Nucleoporin and cleaved caspase-3 from PURPL-WT and PURPL-KO clones with or without DOXO treatment (72 hr). DNA was counterstained with DAPI. Error bars represent SD from 3 experiments. **p<0.01 and ***p<0.001.

Figure 3. Loss of PURPL results in upregulation of basal p53 levels

(A) RT-qPCR analysis for select p53-regulated mRNAs and the housekeeping mRNA SDHA from untreated PURPL-WT and PURPL-KO cells. (B) PURPL-WT and PURPL-KO cells were untreated or treated with DOXO for 24 hr and immunoblotting for p53, p21 and the loading control GAPDH was performed. (C) Immunoblotting was performed from PURPL-WT and PURPL-KO#1 cells transfected for 48 hr with pCB6 or pCB6-PURPL followed by DOXO for 16 hr. GAPDH was used as loading control. (D) PURPL-WT and PURPL-KO#1 cells were transfected for 48 hr with pCB6 or pCB6-PURPL and then treated with Cycloheximide (CHX) for the indicated times; immunoblotting for p53 and the loading control GAPDH was performed. (E) Decay curve for p53 protein quantitated by densitometry is shown for the immunoblot shown in Figures 3D and S9. (F) Immunoblotting for p53 and the loading control GAPDH was performed from PURPL-WT and PURPL-KO RKO, SW48 and DLD1 cells. (G, H) Parental HCT116, RKO, SW48, SK-CO-1, DLD1 and HT29 were transfected for 48 hr with CTL-ASO or PURPL-ASO and the levels of p53 and the loading control GAPDH were assessed by immunoblotting. Error bars represent SD from 3 experiments. *p<0.05 and **p<0.01.

Figure 4. PURPL-deficient cells show reduced proliferation in vitro and impaired tumor growth in vivo

(A) Cell proliferation assays determined by Cell counting Kit-8 at the indicated time points were performed from untreated PURPL-WT and PURPL-KO cells 24 hr after seeding the cells in 96-well plates. (B) HCT116 cells were transfected with a CTL-ASO or PURPL-ASO (50 nM) and cell proliferation assays were performed as described in “(A)”. (C) Untreated PURPL-WT and PURPL-KO cells were seeded at low density in 12-well plates and colony formation assays were performed after 2 weeks. (D) Tumor volume (N=10 mice/each group) in mice was measured by caliper assessment after injecting PURPL-WT and PURPL-KO cells in mice. (E, F) Mice were euthanized after 30 days, tumors were excised and weighed. Average tumor mass at Day 30 is shown (E). Error bars represent SD from 3 experiments in A–C. *p<0.05, **p<0.01 and ***p<0.001.

Figure 5. PURPL associates with MYBBP1A and prevents the formation of a p53-MYBBP1A complex

(A, B) RNA pulldowns were performed using in vitro-transcribed biotinylated PURPL (Bi-PURPL) RNA or biotinylated luciferase (Bi-Luc) RNA and HCT116 whole cell lysates. Associated proteins were pulled down with streptavidin beads and analyzed by SDS-PAGE and mass spectrometry. (A) Peptide spectrum matches (PSMs) corresponding to MYBBP1A in the Bi-Luc and Bi-PURPL pulldowns from mass spectrometry analysis, and (B) spectra, show the four MYBBP1A peptides that associate with PURPL in the RNA pulldowns and mass spectrometry. (C) RNA pulldowns were performed as described in ‘A” followed by immunoblotting for MYBBP1A. (D) The enrichment of PURPL was measured by RT-qPCR from MYBBP1A RNA IPs (RIP) performed from formaldehyde-crosslinked HCT116 cells. IgG IP and the housekeeping SDHA mRNA were used as negative controls. (E) Immunoblotting for MYBBP1A and the loading control GAPDH was performed from PURPL-WT and PURPL-KO HCT116 whole cell lysates. (F) Nucleoplasmic and nucleolar fractions were prepared from HCT116 cells and the levels of the nucleolar pre-rRNA, the nucleoplasmic MALAT1 and PURPL were measured by RT-qPCR. (G) Nucleoplasmic (NPL), nucleolar (NCL) and cytoplasmic (CYT) fractions were prepared from PURPL-WT and PURPL-KO cells and the levels of Histone H3, Nucleolin and Tubulin were assessed as controls for Nucleoplasmic, nucleolar and cytoplasmic fractions, respectively. (H) The interaction of MYBBP1A with p53 was determined by immunoblotting following co-IPs from PURPL-WT and PURPL-KO nucleoplasmic extracts. Lysate refers to whole cell extract prepared from PURPL-WT cells. Error bars represent SD from 3 experiments. **p<0.01.

Figure 6. PURPL associates with MYBBP1A via the adaptor protein HuR

(A) In vivo UV-crosslinked HCT116 cells were subjected to RNA immunoprecipitation (RIP) with anti-MYBBP1A or IgG antibody. The levels of PURPL or the housekeeping SDHA mRNA were measured in the IP material by RT-qPCR. (B) Peptide spectrum matches (PSMs) corresponding to HuR in the Bi-Luc and Bi-PURPL pulldowns from mass spectrometry analysis is shown. (C) Partial sequence of PURPL RNA and the consensus high affinity HuR binding motif (UAUUUAU) at the 3′end is shown. (D) Streptavidin pulldowns were performed using HCT116 whole cell lysates and in vitro transcribed Bi-Luc or Bi-PURPL and the eluted material (undiluted or 1:10 diluted) was subjected to immunoblotting for HuR or the negative control GAPDH. (E) Bi-Luc or Bi-PURPL were incubated with GST or GST-HuR and the mixture was then added to streptavidin beads. Enrichment of GST-HuR in the RNA pulldowns was determined by immunoblotting for HuR. (F) RNA IPs (RIP) assays were performed from UV-crosslinked HCT116 cell lysates using HuR antibody or IgG. Enrichment of PURPL in the IP material was assessed by RT-qPCR. SDHA mRNA was used as negative control. (G) RIP assays using HuR antibody or IgG were performed after sonicating UV-crosslinked HCT116 cell lysates. Enrichment of PURPL-ARE and not the nearby Non-ARE sequence in PURPL in the IP material was assessed by RT-qPCR normalized to GAPDH using primers that span the ARE or Non-ARE region. (H) HuR-MYBBP1A interaction was examined in HCT116 cells by immunoprecipitating MYBBP1A from whole cell lysates followed by immunoblotting for HuR and MYBBP1A. IgG IP was used as control. “IgG-HC” refers to the IgG heavy chain. (I) MYBBP1A was immunoprecipitated from HCT116 whole cell lysates and the enrichment of HuR in the IP material was assessed by immunoblotting in the presence or absence of RNase A. GAPDH was used as negative control. IgG-HC refers to the IgG heavy chain. (J) HCT116 cells were transfected with CTL siRNA or HuR siRNAs for 48 hr and knockdown of HuR was determined by immunoblotting using GAPDH as loading control. (K) RIP assays were performed from formaldehyde-crosslinked HCT116 cell lysates transfected with CTL siRNA or HuR siRNAs. Enrichment of PURPL normalized to GAPDH mRNA in the IP material was assessed by RT-qPCR. (L) Immunoblotting for MYBBP1A was performed for the RIP assays in “K”. Error bars represent SD from 3 experiments. **p<0.01.

Figure 7. Silencing MYBBP1A in PURPL-KO cells partially rescues basal p53 levels and cell proliferation

(A, B) PURPL-WT and PURPL-KO cells were transfected with CTL siRNA or MYBBP1A siRNAs for 48 hr. The levels of MYBBP1A, p53 and the loading control GAPDH were assessed by immunoblotting from whole cell extracts (A) and p21 mRNA levels were measured by RT-qPCR normalized to GAPDH mRNA (B). (C) PURPL-WT and PURPL-KO cells were transfected with CTL siRNA or MYBBP1A siRNAs and cell proliferation was assessed by trypan blue exclusion cell count assay from 2 independent experiments. (D–F) Analysis of PURPL and MYBBP1A mRNA levels in CRC patient samples in the microarrays from the UMMC cohort. PURPL and MYBBP1A mRNA levels (log2 transformed) were compared between normal (N=79) and tumor (N=79) samples (D) and between p53WT (N=42) and mutant p53 tumors (N=37) (E). (F) Significant positive correlation (correlation coefficient shown as “r”) between PURPL and MYBBP1A mRNA levels was observed in the p53WT CRC tumors. “N” represents the number of samples in D–F. Error bars in “B” represent SD from 3 experiments. *p<0.05 and **p<0.01.