PCBP2 Mediates Olaparib Resistance in Breast Cancer by Inhibiting m6A Methylation to Stabilize PARP1 mRNA

Abstract

Base excision repair (BER), a critical pathway for repairing DNA single-strand breaks, is mediated by PARP, which plays a pivotal role in maintaining genomic stability. Targeting PARP with PARP inhibitors (PARPi) has emerged as an effective strategy for treating BRCA-mutated breast cancers characterized by homologous recombination deficiency. However, PARPi resistance remains a major challenge in the treatment of BRCA-mutated breast cancer. Using bioinformatics analysis and cellular-level experiments, we discovered that the RNA-binding protein PCBP2 contributes to resistance to the PARPi olaparib in BRCA-mutated breast cancer by increasing PARP1 expression via interference with the m6A methylation machinery. PCBP2 was upregulated in olaparib-resistant cells, and PCBP2 overexpression in BRCA-mutated breast cancer cells increased resistance to olaparib and enhanced cell proliferation under treatment. Mechanistically, PCBP2 directly interacted with PARP1 mRNA, inhibiting m6A methylation and stabilizing the mRNA. PCBP2-mediated upregulation of PARP1 enhanced DNA repair activity, contributing to olaparib resistance. Together, these findings unveil a mechanism by which PCBP2 upregulates PARP1 to promote olaparib resistance in BRCA-mutated breast cancer, indicating that targeting this pathway could represent a therapeutic strategy to overcome PARPi resistance in breast cancer.

Significance: PCBP2-induced suppression of m6A methylation increases PARP1 to promote DNA damage repair and confer resistance to olaparib in BRCA-mutated breast cancer, making PCBP2 a potential therapeutic target to enhance PARP inhibitor sensitivity.

Graphical abstract

Figure 1.

PCBP2 is associated with olaparib sensitivity. A–D, SUM149PT and HCC1937 cells were stably transduced with LV3 (shNC) or PCBP2 shRNA (shPCBP2) lentiviruses for PCBP2 knockdown experiments. SUM149PT and HCC1937 cells were transduced with LV5 (vector) and LV5-PCBP2 (oePCBP2) lentiviruses for control and PCBP2 overexpression experiments, respectively. Western blotting was used to assess protein levels of PCBP2 and PARP1, and qRT-PCR was performed to measure PCBP2 mRNA levels. E–H, CCK-8 assay was used to measure cell viability in cells treated with olaparib (Ola) for 72 hours. I, Left, clonogenic assays were performed to evaluate the colony-forming ability of SUM149PT cells with or without olaparib treatment (10 μmol/L) for 7–14 days. Right, the number of clones was quantified. J, Left, EdU assays were performed to assess the proliferation of SUM149PT cells following treatment with olaparib (10 μmol/L) or DMSO for 72 hours. Right, percentage of EdU-positive cells was quantified and plotted. Scale bar, 25 μm (high magnification). K, Clonogenic assays were performed as in I using SUM149PT cells overexpressing PCBP2. L, EdU assays were performed as in J using SUM149PT cells overexpressing PCBP2. M, Western blot analysis of PARP1 and PCBP2 protein levels in SUM149PT and HCC1937 cells. Cells were transfected with vector or oePCBP2, followed by siRNA targeting PARP1 (siPARP1) or negative control (siNC). GAPDH was used as the loading control. N and O, CCK-8 assay was used to measure cell viability in cells treated with olaparib for 72 hours. P–S, Whole cell lysate (WCL) and chromatin-binding protein (CHR) were extracted and analyzed by Western blotting with the indicated antibodies. Data are presented as the mean ± SEM (n = 3). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

Stable knockdown of PCBP2 enhances olaparib sensitivity both in vitro and in vivo in olaparib-resistant breast cancer cells. A, BRCA-mutated cell lines SUM149PT and HCC1937 were subjected to a gradual increase in the concentration of olaparib to develop acquired resistance. The IC₅₀ values of olaparib-resistant SUM149PT^OlaR and HCC1937^OlaR, as well as the original parental (Par) cell lines, were determined by CCK-8 assay. B, Talazoparib IC₅₀ curves of parental SUM149PT and cells with acquired resistance to olaparib. C, Veliparib IC₅₀ curves of parental SUM149PT and cells with acquired resistance to olaparib. D, Niraparib IC₅₀ curves of parental SUM149PT and cells with acquired resistance to olaparib. E and F, Western blotting was used to assess protein levels of PCBP2 and PARP1, and qRT-PCR was performed to measure PCBP2 mRNA levels. G and H, SUM149PT^OlaR and HCC1937^OlaR cells were stably transduced with shNC and shPCBP2 lentiviruses. Western blotting was used to assess protein levels of PCBP2 and PARP1, and qRT-PCR was performed to measure PCBP2 mRNA levels. I, CCK-8 assay was used to measure cell viability in cells treated with olaparib for 72 hours. J, Left, EdU assays were used to characterize the proliferation of SUM149PT^OlaR and HCC1937^OlaR cells treated with olaparib. Right, percentage of EdU-positive cells was quantified. Cells were treated with 10 μmol/L olaparib for SUM149PT^OlaR and 20 μmol/L olaparib for HCC1937^OlaRfor 72 hours. Scale bar, 25 μm (high magnification). K, Left, clonogenic assays were conducted to assess the colony-forming ability of SUM149PT^OlaR and HCC1937^OlaR cells in the presence of olaparib. Cells were treated with 10 μmol/L olaparib for SUM149PT^OlaR and 20 μmol/L olaparib for HCC1937^OlaR for 7–14 days. Right, the number of colonies was quantified. L and M, Whole cell lysate (WCL) and chromatin-binding protein (CHR) were extracted and analyzed by Western blotting with the indicated antibodies. N, Schematic representation of the PCBP2 knockdown xenograft breast cancer mouse model using SUM149PT^OlaR cells and the treatment regimen. O, At the end of treatment, tumors were excised and photographed. P, Tumor size was measured at the indicated time intervals and calculated, and growth curves were plotted using average tumor volume within each experimental group at the designated time points. Q, Tumor weights. R, Representative IHC images of PCBP2, PARP1, and γH2AX in subcutaneous tumors. Scale bar, 50 μm (high magnification). Data are presented as the mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

PCBP2 upregulates PARP1 by directly binding to the PARP1 mRNA 3′ UTR. A, Pearson pairwise correlation plot of PCBP2 and PARP1 mRNA expression obtained from the bc-GenExMiner database using RNA sequencing analysis. B–E, qRT-PCR was performed to measure PARP1 mRNA levels. F, RIP assay for the interaction between PCBP2 and PARP1 mRNA in SUM149PT and HCC1937 cells, showing enrichment of PARP1 mRNA, as measured by qRT-PCR. RIP with nonspecific IgG was set as control. Western blot of PCBP2 showing equal amount of input PCBP2 protein in the two groups. G, Schematic structures of PCBP2 and deletion mappings of PCBP2. Vec, vector; FL, full length PCBP2; T, truncated fragment of PCBP2, with T1: 1–96; T2: 76–286; and T3: 163–365. H, RIP-qPCR, using full-length or truncated PCBP2 protein to identify the PARP1 mRNA–binding domains in PCBP2. I, RNA pulldown assay using a PARP1 mRNA 5′ UTR probe, coding sequence (CDS) probe, or 3′ UTR probe in SUM149PT and HCC1937 cells. PCBP2 protein was characterized by Western blotting in the indicated precipitates of the pulldown assay. J, Western blot analysis of HA-tagged PARP1 lacking the 3′ UTR [oePARP1(Δ3′ UTR)] in SUM149PT and HCC1937 cells transduced with shNC or shPCBP2. Cells were cotransfected with either vector or oePARP1(Δ3′ UTR) construct. K and L, Expression of PARP1 mRNA, measured by qRT-PCR, in cells transduced with the indicated lentiviruses and treated with α-amanitin for 0, 6, 12, 18, 24, and 30 hours. Data are presented as the mean ± SEM (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

Depletion of PCBP2 facilitates m⁶A methyltransferase complex recruitment and accelerates PARP1 mRNA degradation. A, Top, predicted m⁶A site information was obtained from the WHISTLE database. Left, location of the PCBP2 binding site and a potential m⁶A site in the PARP1 mRNA 3′ UTR. Right, abundance of the PARP1 transcript in mRNA immunoprecipitated with an anti-m⁶A antibody was measured by qRT-PCR and normalized to IgG. B and C, The mRNA level of PARP1 was determined by qRT-PCR after transfection with the indicated siNC or siRNAs. D–I, METTL3, METTL14, and WTAP were immunoprecipitated, followed by qRT-PCR for assessing the association of PARP1 mRNA with the indicated m⁶A methyltransferase after overexpression or knockdown of PCBP2. RIP with nonspecific IgG was set as the control. J and K, Abundance of PARP1 mRNA, as measured by qRT-PCR, among mRNA immunoprecipitated with anti-m⁶A antibody from cells transduced with the indicated vectors. L, PARP1 mRNA 3′ UTR, either wild-type or with a mutated m⁶A consensus sequence (A–G), was cloned into a dual-luciferase reporter. M and N, Luciferase activity of the reporter gene with a PARP1 mRNA 3′ UTR was measured and normalized to Renilla luciferase activity in cells after transduction with shPCBP2, oePCBP2, or their corresponding empty vectors. O–Q, Luciferase activity was measured after cotransfection with shPCBP2 and siMETTL3/siMETTL14/siWTAP. R and S, Relative expression of PARP1 mRNA in cells cotransfected with shPCBP2 and either siNC or siMETTL3, as measured by qRT-PCR after α-amanitin treatment. T, Schematic illustration of the targeted RNA methylation system. U, Abundance of PARP1 among mRNA immunoprecipitated with anti-m⁶A antibody from cells transfected with the indicated vectors, as measured by qRT-PCR. V, Protein levels of PARP1 and PCBP2 were measured by Western blotting after transfection with the indicated vectors. W, Relative expression of PARP1 mRNA in cells transfected with the indicated vectors, as measured by qRT-PCR after α-amanitin treatment. X, At the end of treatment, tumors were excised and imaged. Data are presented as the mean ± SEM (n = 3). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

YTHDF2-dependent regulation of PARP1 mRNA stability modulates olaparib sensitivity. A–D, The mRNA level of PARP1 was determined by qRT-PCR after transfection with siNC or siYTHDF1/2. E–J, YTHDF1/2 was immunoprecipitated, followed by qRT-PCR for assessing the association of PARP1 mRNA with YTHDF1/2 after overexpression or knockdown of PCBP2. RIP with nonspecific IgG was set as control. Western blot of YTHDF1/2 showed equal amount of input YTHDF1/2 protein in the two groups. K and L, Luciferase activity was measured after cotransfection with shPCBP2 and siYTHDF2. M and N, Relative expression of PARP1 mRNA in cells transduced with shPCBP2 and cotransfected with either siNC or siYTHDF2, as measured by qRT-PCR after α-amanitin treatment. O, Schematic illustration of the targeted RNA methylation system. P and Q, qRT-PCR was performed to measure PARP1 mRNA levels. R, Protein level of PARP1 was measured by Western blotting after transfection with the indicated vectors. S, Relative expression of PARP1 mRNA in cells transfected with the indicated vectors, as measured by qRT-PCR after α-amanitin treatment. Data are presented as the mean ± SEM (n = 3). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Clinical relevance of the PCBP2–m⁶A–PARP1 drug resistance axis in patients with breast cancer. A, Representative IHC staining of PCBP2, PARP1, γH2AX, METTL3, and YTHDF2 in breast cancer patient samples categorized by clinical response to PARPis (olaparib-sensitive vs. olaparib-resistant). Scale bar, 50 μm (high magnification). B, Quantitative IHC analysis of PCBP2, PARP1, γH2AX, METTL3, and YTHDF2 expression in samples of patients with breast cancer (n = 10, 5 olaparib-sensitive and 5 olaparib-resistant). Patients with olaparib resistance showed significantly higher PCBP2 and PARP1 expression and lower γH2AX levels compared with sensitive patients, whereas METTL3 and YTHDF2 expression showed no significant differences. Data are presented as the mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01.