Abstract
Poly(ADP-ribose) polymerase 1 (PARP1) is essential for the progression of several types of cancers. However, whether and how PARP1 is stabilized to promote genomic stability in triple-negative breast cancer (TNBC) remains unknown. Here, we demonstrated that the deubiquitinase USP15 interacts with and deubiquitinates PARP1 to promote its stability, thereby stimulating DNA repair, genomic stability and TNBC cell proliferation. Two PARP1 mutations found in individuals with breast cancer (E90K and S104R) enhanced the PARP1–USP15 interaction and suppressed PARP1 ubiquitination, thereby elevating the protein level of PARP1. Importantly, we found that estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) inhibited USP15-mediated PARP1 stabilization through different mechanisms. ER bound to the USP15 promoter to suppress its expression, PR suppressed the deubiquitinase activity of USP15, and HER2 abrogated the PARP1–USP15 interaction. The specific absence of these three receptors in TNBC results in high PARP1 levels, leading to increases in base excision repair and female TNBC cell survival.
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Data availability
The human breast cancer genomic data were derived from the CPTAC Research Network at https://cptac-data-portal.georgetown.edu/study-summary/S015. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank CPTAC and TCGA for providing high-quality high-throughput data for public analysis. We thank A. Geng, H. Zhang, W. Zhang and Z. Zhen for critically reading the manuscript and helpful discussions. This work was supported by the National Key R&D Program of China (grant numbers 2022YFA1103703 and 2021YFA1102003 to Z.M.) and the National Natural Science Foundation of China (grant numbers 81972457 and 32171288 to Y.J. and 82225017, 32270750 and 82071565 to Z.M.).
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X.S., H.T. and Y.C. performed experiments, analyzed data and wrote the manuscript. Z. Chen, Z.H., Z. Cui, Y.T., J.Y., Y.F., Z.Z., Q.H., Q.L., X.X. and X.W. were involved in data collection. Y.J. and Z.M. were involved in the conception and design, data interpretation, manuscript writing and final approval of the manuscript.
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Nature Cancer thanks Joaquin Arribas, Luca Busino, and the other, anonymous, reviewers for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 USP15 interacts with PARP1 in a PAR-independent manner.
(a) Screening of putative PARP1-interacting DUBs using 60 vectors encoding different DUBs. Vectors encoding HA-tagged DUBs and GFP-tagged PARP1 were cotransfected into HEK293 cells. After 24 h, cells were harvested for a co-IP assay with beads coated with GFP nanobodies prior to Western blot analysis with the indicated antibodies. (b, c) Analysis of PARP1 protein levels in USP1- or USP21-depleted cells treated with cycloheximide (CHX). HEK293 cells with depletion of USP1 (b) or USP21 (c) were treated with CHX at a concentration of 100 μg/mL and harvested for Western blot analysis at the indicated time points. (d) PARP1 mRNA levels did not change in USP15-depleted cells treated with CHX. HEK293 cells with USP15 depletion were treated with CHX at a concentration of 100 μg/mL and harvested for total RNA extraction and quantitative PCR experiments. n = 3 technical replicates. (e-f) USP15 interacted with PARP1 in a PAR-independent manner. HEK293 cells were treated with the PARP1 inhibitors 1 μM olaparib (e) or veliparib (f) for 24 h before being harvested for IP and Western blot analysis. (g) The catalytically dead PARP1 mutant retained its interaction with USP15. Plasmids encoding Flag-tagged PARP1-WT or PARP1-E988K were transfected into HEK293 cells and co-IP experiments was performed with magnetic beads coated with anti-Flag antibodies, and Western blot analysis was performed with the indicated antibodies. (h) Analysis of the interaction between USP15 and PARP1 on chromatin or in nucleoplasm. All experiments were repeated three times independently with similar results; data of one representative experiment are shown.
Extended Data Fig. 2 USP15 stabilizes PARP1 to regulate BER.
(a) Cells with USP15 depletion were transfected with a vector encoding ubiquitin and the control vector, USP15-WT or the catalytically dead USP15-C269A mutant. Cells were supplemented with 10 μM MG132 for 12 h, and co-IP experiments with antibodies against PARP1 were performed 24 h post transfection prior to Western blot analysis with the indicated antibodies. (b) USP15 deubiquitinated PARP1 in vitro. (c) The identification of E3 ubiquitin ligases that modify PARP1 after USP15 loss. The indicated E3 ubiquitin ligases were knocked down in USP15-depleted HEK293 cells, and co-IP experiments were then performed with an antibody against PARP1, followed by Western blot analysis against K48-linked ubiquitin. (d) Analysis of RNF144A, IDUNA, RNF168, CHFR and TRIP12 levels in the indicated factor depleted HEK293 cells. (e) Co-IP analysis of the PARP1-USP15 interaction in MMS-treated HEK293 cells. HEK293 cells were treated with MMS at a concentration of 500 μM and incubated for 2 h before being harvested for co-IP with an antibody against PARP1. The precipitated proteins were then further treated with λ phosphatase prior to Western blot analysis with the anti-USP15 antibody. (f) Co-IP analysis of the PARP1-USP15 interaction in HEK293 cells treated for 6 h with MMS at different concentrations. Cells were harvested for co-IP with an antibody against PARP1. The precipitated proteins were then further treated with λ phosphatase prior to Western blot analysis with the anti-USP15 antibody. (g) Western blot analysis of the PARP1 protein level in control with USP15-depleted HEK293 cells. Cells were treated with MMS at the indicated concentrations for 6 hours before being harvested for Western blot analysis. Data are expressed as mean ± s.d., n = 3 independent biological samples. (h) Analysis of H2AX PARylation in MMS treated control and USP15 depleted cells. HEK293 cells were treated with MMS at a concentration of 400 μM and incubated for 6 h before being harvested for co-IP experiments with an antibody against PAR. All experiments were repeated three times independently with similar results; data of one representative experiment are shown.
Extended Data Fig. 3 PARP1 protein levels positively correlate with USP15 in TNBC.
(a) The Hematoxylin and eosin (H&E) staining analysis of the breast cancer tissue specimens, and the associated normal tissue samples. (b) Western blot analysis of PARP1 and USP15 expression in 14 other sets of TNBC tissues and the corresponding adjacent normal mammary tissues (4 of these tissues are shown in Fig. 3c). (c) Western blot analysis of PARP1 and USP15 expression in the other 12 sets of non-TNBC tissues and the corresponding adjacent normal mammary tissues (4 of these tissues are shown in Fig. 3e). (d–e) The comparison of PARP1 or USP15 protein levels between 90 sets of breast cancer tissues, namely, 54 TNBC tissues and 36 non-TNBC tissues, and the corresponding adjacent normal mammary tissues by the immunohistochemistry assay. Data are expressed as mean ± s.e.m.. (f) Representative immunohistochemistry images of USP15 and PARP1 expression in breast cancer tissues and adjacent normal mammary tissues. (g) Comparison of PARP1 mRNA levels between TNBC tissues and the corresponding adjacent normal mammary tissues. mRNA was extracted from clinical tissues and analyzed by quantitative PCR, n = 3 technical replicates. P values are indicated (d–e), and statistical significance was determined by Student’s t test. Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a–c, f–g).
Extended Data Fig. 4 USP15 interacts with and deubiquitinates PARP1 in TNBC cells.
(a) Co-IP analysis of the PARP1-USP15 interaction in different cell lines. (b) USP15 deubiquitinated PARP1 in MDA-MB-231 cells. (c) PARP1 mRNA levels did not change in USP15-depleted MDA-MB-231 cells, n = 3 technical replicates. (d, e) Immunofluorescence analysis of PARP1 in control and USP15-depleted MDA-MB-231 cells. And quantitative analysis was performed with ImageJ software. n = 20 for each group. Representative images are shown in (e). (f, g) Immunofluorescence analysis of PAR in control or USP15-depleted MDA-MB-231 cells. Cells were treated with MMS prior to immunostaining with the antibody against PAR. And quantitative analysis was performed with ImageJ software. n = 20 for each group. Representative images are shown in (g). (h) Analysis of BER efficiency in MDA-MB-231 cells with USP15 and/or PARP1 depletion. n = 3 independent biological samples. (I, j) Analysis of PARP1 and USP15 levels in PARP1-KO, USP15-KO, or PARP1 & USP15 both KO (dKO) MDA-MB-231 cells. (k, l) Analysis of PARP1 and USP15 levels in different groups of MDA-MB-231 cells. (m, n) Analysis of genomic stability in MDA-MB-231 cells in the presence or absence of 200 μM MMS by an alkaline comet assay. At least 50 cells per group were included for analysis. (o, p) Analysis of PARP1 and USP15 levels in different groups of HCC70 cells. (q, r) Analysis of genomic stability in HCC70 cells in the presence or absence of 200 μM MMS by an alkaline comet assay. Different groups of cells as indicated were treated with 200 μM MMS or DMSO and were then harvested for the alkaline comet assay. At least 50 cells per group were included for analysis. All experiments were repeated at least three times. Data are expressed as mean ± s.d. (h). Data are expressed as the mean ± s.e.m., each point represents a cell, the cells used for analysis in each experiment were from a single replicate. (d, f, m-n, q-r). n.s., not significant. P values are indicated (d, f, h, m-n, q-r). Statistical significance was determined by Student’s t test (D, F), or one-way ANOVA test followed by Tukey’s multiple comparison (h, m-n, q-r). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a-c, h-p).
Extended Data Fig. 5 USP15-PARP1 axis promotes BER to stabilize genomic stability in TNBC cells.
(a, b) Analysis of genomic stability in MDA-MB-231 cells in the presence or absence of 200 μM MMS by a neutral comet assay. At least 50 cells per group were included for analysis. (c, f) Analysis of the survival of MDA-MB-231 (c, d) and HCC70 (e, f) cells in response to MMS. Different groups of cells were treated with MMS at the indicated concentrations, and a clonogenic assay was performed to calculate the survival rates. n = 3 independent biological samples. (g) The changes in the numbers of HCC1937 cells in different groups. The different groups of BRCA1-deficient HCC1937 cells were plated in 96-well plates at a density of 300 cells/well and counted at 24 h, 48 h, 72 h and 96 h post splitting. n = 3 independent biological samples. (h) The changes in the numbers of MDA-MB-436 cells in different groups. n = 3 independent biological samples. (i-k) Analysis of PARP1 and USP15 levels in different groups of cells: (i, j) HCC1937, (k) MDA-MB-436. (l, m) Analysis of the survival of MDA-MB-231 (l) and HCC70 (m) cells in response to the PARP1 inhibitor olaparib. Different groups of cells were treated with olaparib at the indicated concentrations, and a clonogenic assay was performed to calculate the survival rates. The cells with BRCA1 depletion were used as the positive control. n = 3 independent biological samples. (n, o) Analysis of BRCA1 level in BRCA1-depleted MDA-MB-231 (n) and HCC70 (o) cells. Data are expressed as mean ± s.d. (c-h, l-m). data are expressed as mean ± s.e.m., each point represents a cell, the cells used for analysis in each experiment were from a single replicate. (a-b). n.s., not significant. P values are indicated (a-b, g-h, l-m). Statistical significance was determined by Student’s t test (l-m), or one-way ANOVA test followed by Tukey’s multiple comparison (a-b, g-h). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (c-o).
Extended Data Fig. 6 USP15-PARP1 axis promotes BER to accelerate TNBC growth.
(a) Analysis of PARP1 and USP15 levels in different groups of HCC70 cells. (b, c) Analysis of MDA-MB-231 xenograft growth in vivo. Different groups of MDA-MB-231 cells were engrafted into the right flank of immunocompromised mice. At different time points post injection, tumors were measured, and tumor volumes were calculated and recorded. Tumor volumes are shown in (b). Tumor weights at the experimental endpoint are shown in (c). A representative picture of the tumors is shown in (c). Data are expressed as mean ± s.e.m., n = 5 mice for each group. (d) Analysis of 8-oxoguanine and γH2AX positive cells in MDA-MB-231 xenograft samples. Data are expressed as mean ± s.e.m., n = 5 for each group, each point represents a xenograft tumor, the tumors used for analysis in each experiment were from a single replicate. n.s., not significant. P values are indicated (b-d). Statistical significance was determined by one-way ANOVA test followed by Tukey’s multiple comparison (c-d), or two-way ANOVA test followed by Tukey’s multiple comparison (b). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a).
Extended Data Fig. 7 PARP1 mutations promote USP15-mediated PARP1 stabilization.
(a) Bioinformatic analysis of basal-like breast cancer patients carrying mutations in USP15 and PARP1. (b) Analysis of the interactions between PARP1 and WT or mutant USP15. (c) Generation of MDA-MB-231 cell lines harboring endogenous mutations in PARP1 genes via CRISPR-Cas9 technology. (d) Co-IP analysis of the PARP1-USP15 interaction in the PARP1 PARP1+/+, PARP1+/E90K, PARP1+/S104R MDA-MB-231 cells. (e) Analysis of the K48-linked ubiquitination on PARP1 in PARP1+/+, PARP1+/E90K, PARP1+/S104R MDA-MB-231 cells. (f) Analysis of BER efficiency in PARP1 PARP1+/+, PARP1+/E90K, PARP1+/S104R MDA-MB-231 cells. n = 3 independent biological samples. (g) Analysis of genomic stability in PARP1+/+, PARP1+/E90K, PARP1+/S104R MDA-MB-231 cells with USP15 depleted in the absence or presence of 200 μM MMS using an alkaline comet assay. At least 50 cells per group were included for analysis. (h) Analysis of the enzymatic activity of PARP1 WT, E90K and S104R in vitro. (i) Analysis of DNA binding ability of PARP1 WT, E90K and S104R by EMSA assay. (j, k) Analysis of recruitment of WT, E90K and S104R PARP1 to DNA damage sites in U2OS cells following microirradiation. n = 8 for each group were quantified using Leica Las X. Representative images are shown in (k). (l-o) PARP1 E90K or S104R mutants significantly suppressed MMS-induced apoptosis in MDA-MB-231 cells. n = 3 independent biological samples. (p) Analysis of PARP1 trapping on chromatin in PARP1 WT and mutant cells. (q–s) Analysis of xenograft growth in vivo. The PARP1-KO HCC70 cells were infected with lentiviruses bearing vectors expressing PARP1 WT, E90K or S104R. These cell lines were further depleted USP15 with shRNA. Tumor volumes are shown in (r). Tumor weights at the experimental endpoint are shown in (S). A representative picture of the tumors is shown in (s). Data are expressed as mean ± s.e.m., n = 5 mice for each group. (t) Analysis of PARP1 and USP15 levels in different groups of HCC70 cells. (u–w) Analysis of xenograft weight. The PARP1-KO HCC70 cells were infected with lentiviruses bearing vectors expressing PARP1 WT, E90K or S104R. These cell lines were further chromosomally integrated with a control vector or a vector expressing USP15. Cells were engrafted into immunocompromised mice, and mice were treated with DMSO or olaparib. Data are expressed as mean ± s.e.m., n = 5 mice for each group. Data are expressed as mean ± s.d. (f, l, n). Data are expressed as mean ± s.e.m., each point represents a cell, the cells used for analysis in each experiment were from a single replicate (g, j). n.s., not significant. P values are indicated (f-g, j, l, n, r-s, u-w). Statistical significance was determined by Student’s t test (f, j, l, n), or one-way ANOVA test followed by Tukey’s multiple comparison (g, s, u-w), or two-way ANOVA test followed by Tukey’s multiple comparison (r). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (b, d-f, h-i, l, n, p-q, t).
Extended Data Fig. 8 ER/PR/HER2 negatively regulate USP15 and PARP1 expression.
(a, b) Comparison of the changes in the PARP1 (a) and USP15 (b) protein levels in tumor tissues and the corresponding adjacent normal mammary tissues between the TNBC and non-TNBC groups. The analysis was based on Fig. 3. n = 16 for non-TNBC and n = 18 for TNBC. (c) Analysis of USP15 and PARP1 level in TNBC cell lines and non-TNBC cell lines. (d) Analysis of BER efficiency in TNBC cell lines and non-TNBC cell lines. n = 3 independent biological samples. (e, f) Comparison of the changes in the PARP1 expression and BER efficiency between cells of TNBC group and non-TNBC group, n = 4 cell lines. (g) Analysis of ER, PR and HER2 protein levels in MDA-MB-231 cells transfected with different groups of vectors. Cells were harvested for Western blot analysis at 24 h post transfection. (h) Analysis of USP15 and PARP1 protein levels in MDA-MB-231 and HCC70 cells overexpressing ER, PR and HER2. (i) Analysis of USP15 and PARP1 protein levels in MDA-MB-231 cells overexpressing ER, PR or HER2. (j) Analysis of PARP1 trapping on chromatin in ER, PR and HER2-overexpressing cells. (k, l) Analysis of xenograft growth in vivo. The HCC70 cells with a control vector or vectors expressing ER, PR and HER2 were engrafted into immunocompromised mice, and mice were treated with DMSO or olaparib. n = 6 mice for the group of pControl+olaparib, and n = 5 mice for other groups. Tumor volumes are shown in (k). Tumor weights at the experimental endpoint are shown in (l). (m) Analysis of USP15 and PARP1 protein levels in MDA-MB-436 cells overexpressing ER, PR and HER2. n = 3 independent biological samples. (n) Analysis of USP15 and PARP1 protein levels in HCC1954, MCF7 and T47D cells overexpressing ER, PR or HER2. Data are expressed as mean ± s.d. (d, m). Data are expressed as mean ± s.e.m. (a-b, e-f, k-l). P values are indicated (a-b, d-f, k-l). Statistical significance was determined by Student’s t test (a-b, d-f), or one-way ANOVA test followed by Tukey’s multiple comparison (l), or two-way ANOVA test followed by Tukey’s multiple comparison (k). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (c-d, g-j, m-n).
Extended Data Fig. 9 ER/PR/HER2 regulate USP15-PARP1 axis via distinct mechanisms.
(a) Analysis of the PARP1 mRNA level in MDA-MB-231 cells overexpressing ER, n = 3 technical replicates. (b) Comparison of the USP15 mRNA level between TNBC tissues and adjacent normal mammary tissues, n = 3 technical replicates. (c) Comparison of the USP15 mRNA level between non-TNBC tissues and adjacent normal mammary tissues, n = 3 technical replicates. (d) Cells transfected with vectors encoding EGFP or PR were supplemented with 10 μM MG132 for 12 h, then subjected to co-IP experiments. (e) Analysis of USP15 enzymatic activity in the presence or absence of PR using a Ub-CHOP2-reporter deubiquitylation assay. The procedure was previously described (Lim et al.,58). n = 3 independent biological samples. (f) The analysis of the interaction between USP15 and PR in MDA-MB-231 cells using the PLA assay. (g) Analysis of the PARP1-USP15 interaction in the presence or absence of HER2 using the BiFC assay. n = 3 independent biological samples. (h) Analysis of the interaction between HER2 and USP15 in MDA-MB-231 cells. (i) HER2 did not interact with USP15 in vitro. (j) Analysis of HER2 subcellular localization in HCC1954 cells using immunostaining. Cells were transfected with control siRNA or siRNA against HER2 prior to immunofluorescence staining with an anti-HER2 antibody. n = 12 cells for each group. (k) Western blot analysis of HER2 in the cytoplasm and nucleus in BT474 and HCC1954 cells. Cytosolic and nuclear proteins were extracted as previously described (Liu et al.,58). (l) Co-IP assay of the interaction between PARP1 and HER2 in nucleus. (m) The analysis of the interaction between PARP1 and HER2 in HCC1954 cells using the PLA assay. Cells were treated with DMSO or 500 mM MMS for 2 h before PLA assay. Data are expressed as mean ± s.d. (e, g). Data are expressed as mean ± s.e.m., each point represents a cell, the cells used for analysis in each experiment were from a single replicate (j). n.s., not significant. P values are indicated (e, g, j). Statistical significance was determined by student’s t test (g, j), or two-way ANOVA test followed by Tukey’s multiple comparison (e). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a-i, k-m).
Extended Data Fig. 10 The model showing the mechanism by which ER/PR/HER2 suppress USP15-dependent stabilization of PARP1 in triple-negative breast cancer.
ER, PR and HER2 negatively regulate USP15-mediated PARP1 stabilization through different regulatory mechanisms. ER binds to the region from −2345 bp to −2341 bp in the USP15 promoter to suppress USP15 expression, PR inhibits the enzymatic activity of USP15, while HER2 abrogates the interaction between PARP1 and USP15 by competing with USP15 for binding to PARP1. Loss of ER, PR and HER2 in TNBC tissues aberrantly abolished the negative regulation of USP15-mediated PARP1 stabilization, thereby promoting the genomic stability and survival of TNBC cells.
Supplementary information
Supplementary Information
Supplementary Fig. 1. Gating strategy.
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Sun, X., Tang, H., Chen, Y. et al. Loss of the receptors ER, PR and HER2 promotes USP15-dependent stabilization of PARP1 in triple-negative breast cancer. Nat Cancer 4, 716–733 (2023). https://doi.org/10.1038/s43018-023-00535-w
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DOI: https://doi.org/10.1038/s43018-023-00535-w
