CTDP1 regulates breast cancer survival and DNA repair through BRCT-specific interactions with FANCI - PubMed (original) (raw)
CTDP1 regulates breast cancer survival and DNA repair through BRCT-specific interactions with FANCI
Wen-Feng Hu et al. Cell Death Discov. 2019.
Abstract
BRCA1 C-terminal domains are found in a specialized group of 23 proteins that function in the DNA damage response to protect genomic integrity. C-terminal domain phosphatase 1 (CTDP1) is the only phosphatase with a BRCA1 C-terminal domain in the human proteome, yet direct participation in the DNA damage response has not been reported. Examination of the CTDP1 BRCA1 C-terminal domain-specific protein interaction network revealed 103 high confidence interactions enriched in DNA damage response proteins, including FANCA and FANCI that are central to the Fanconi anemia DNA repair pathway necessary for the resolution of DNA interstrand crosslink damage. CTDP1 expression promotes DNA damage-induced FANCA and FANCD2 foci formation and enhances homologous recombination repair efficiency. CTDP1 was found to regulate multiple aspects of FANCI activity, including chromatin localization, interaction with γ-H2AX, and SQ motif phosphorylations. Knockdown of CTDP1 increases MCF-10A sensitivity to DNA interstrand crosslinks and double-strand breaks, but not ultraviolet radiation. In addition, CTDP1 knockdown impairs in vitro and in vivo growth of breast cancer cell lines. These results elucidate the molecular functions of CTDP1 in Fanconi anemia interstrand crosslink repair and identify this protein as a potential target for breast cancer therapy.
Keywords: Breast cancer; Protein-protein interaction networks.
Conflict of interest statement
Conflict of interestThe authors declare that they have no conflict of interest.
Figures
Fig. 1. CTDP1 BRCT domain TAP-MS identifies interactions with Fanconi anemia proteins.
a Representation of CTDP1 and its modular domain organization. The BRCT region cloned for TAP–MS experiments is indicated between amino acids 619–738. b Workflow diagram of the TAP–MS experiment. c SAINT output for Bayesian false discovery rate (SAINT-BFDR) was used to create a high confidence interaction list. Graph represents the fold change in protein representation between CTDP1 BRCT purification and control and the number of proteins at binned intervals of 0.5 SAINT-BFDR scores. d TAP–MS experiments generate a comprehensive interaction network of 103 proteins interacting with the CTDP1 BRCT domain. Red outlined nodes indicate proteins with GO annotations for DNA damage (GO:0006281) and DNA replication (GO:0006260). Edge color represents source of protein interactions (gray: TAP-MS, blue: BisoGenet). e KEGG pathway enrichment determined by ClueGO of the 103 CTDP1 interacting proteins identified by TAP–MS, excluding CTDP1 itself. The most significant term for each cluster is presented in bold font and term _p_-value corrected with Bonferroni step down is presented in parentheses. Threshold for visualization was _p_-value ≤ 0.05 and represented inversely proportional to node size. Exact _p_-values are displayed under most significant group node determined by two-sided hypergeometric test corrected using Bonferroni step down method. f Specificity of protein interactions with the CTDP1 BRCT domain in comparison to 27 other BRCT domain interaction datasets. FANCI and FANCA passing SAINT-BFDR cutoff of ≤0.05 are represented as red circles. g Table detailing central FA pathway proteins’ (FANCA, FANCI, and FANCD2) average spectrum abundance (Ave. Spec.), peptide coverage of protein sequence (Percent Coverage), and SAINT BFDR from TAP-MS. h Analysis of CTDP1 and FANCI expression in the input and immunoprecipation (IP) of control (IgG) or endogenous CTDP1 in complex with FANCI from untreated 293FT cells. i Top: Whole cell lysate (WCL) blot of GFP-FANCI and CTDP1-DDK constructs co-overexpressed in 293FT cells with and without 0.1 μM MMC treated for 24 h. Bottom: IP of DDK-tagged CTDP1 co-immunoprecipitates GFP-tagged FANCI independent of MMC treatment
Fig. 2. Overexpression of CTDP1 regulates FANCI SQ motif phosphorylation at S556 and S559 and chromatin localization.
a FANCI SQ motif phosphorylation at S556 (pS556) and activation of ATM and ATR was evaluated with the indicated antibodies in 293FT cells transfected with control empty vector (EV) or CTDP1-DDK-tagged constructs in the presence and absence of DNA damage with 0.1 μM MMC for 16 h. b FANCI SQ motif phosphorylation at S556 (pS556) upon CTDP1 knockdown and MMC treatment. c Wild-type (WT), phosphatase-dead (D302K), and cancer mutants (R270Q and V705M) constructs of CTDP1 were expressed in 293FT cells treated with MMC, and the impact on SQ motif phosphorylation sites at S556 and S559 were evaluated by western blot with the indicated antibodies. d FANCI phosphorylation at S556 was evaluated with and without 0.4 μM MMC in MDA-MB-231 cells transfected with either EV or DDK-CTDP1 expression constructs. e shScr and shCTDP1 HCT116 cells were treated with or without 100 µM melphalan for 6 h and subjected to cellular fractionation and both CTDP1 and FANCI localization to chromatin was determined by western blot. Total H2AX and tubulin are included as fractionation control for chromatin and soluble protein fractions. Short exposure (S.E.). Long exposure (L.E.) f FANCI association with the DNA damage marker γ-H2AX was evaluated by immunoprecipitation of FANCI (IP:FANCI) followed by western blot for γ-H2AX 293FT cells either untreated or treated with 50 μM cisplatin for 16 h. Dashed line in the CTDP1 blot indicates the image has only been modified to maintain the correct order of samples
Fig. 3. CTDP1 promotes homologous recombination DNA repair.
a Schematic of the DR-GFP reporter homologous recombination assay. b Expression of transfected CTDP1-DDK in HeLa-DR cells. c HR efficiency of transfected cells validated in panel b was measured by flow cytometry quantitation of GFP-positive cells. n = 3. Data represented as mean ± standard error of the mean (SEM); paired two-tailed Student’s _t_-test. d Knockdown of CTDP1 (shCTDP1) and non-targeting scrambled shRNA control (shScr) in HeLa-DR cells evaluated by western blot. e HR efficiency in response to CTDP1 knockdown from cells validated in panel d was measured by flow cytometry quantitation of GFP-positive cells. n = 3; mean ± SEM; paired two-tailed Student’s _t_-test. f Confirmation of protein expression knock down in shScr, shCTDP1, shFANCA, and shBRCA2 targeted cells by western blot for the indicated proteins. g HR efficiency was measured by flow cytometry quantitation of GFP-positive cells in the cells validated in panel f. n = 3; mean ± SEM; paired two-tailed Student’s _t_-test
Fig. 4. Impaired CTDP1 expression in response to DNA damage does not impact ATM activation.
a Western blot analysis of CTDP1 expression profiles across a panel of seven breast cell lines with and without treatment of 100 µM melphalan for 6 h. b λ-phosphatase treatment of immunoprecipitated CTDP1 (IP: CTDP1) in T-47D cells either untreated or treated with 100 μM melphalan for 6 h. c MCF-10A breast cells were treated with or without 100 μM melphalan in the presence or absence of 50 μM proteasome inhibitor MG132 pretreatment for 6 h. Cell lysates were analyzed by western blot for CTDP1 and β-actin protein expression. d Western blot of MCF-10A lysates for CTDP1, total and phospho-ATM (pS1981), pSQ/pTQ motifs, and Tubulin collected at the indicated times following treatment with 100 μM melphalan. e Western blot analysis of the indicated proteins in MCF-10A cells expressing shScr control or shCTDP1 with and without treatment of 100 μM melphalan for 6 h
Fig. 5. CTDP1 knockdown impairs FANCA and FANCD2 DNA damage-induced foci.
a Western blot demonstrating knockdown of CTDP1 and FANCA using targeted shRNAs in MCF-10A cells. b Representative immunofluorescence of FANCA alone and merged with DAPI (white and blue color, respectively) in MCF-10A cells expressing the indicated shRNAs (shScr, shCTDP1, or shFANCA) treated with 0.2 μM MMC for 24 h. Scale = 5 μm. c Graph representing the percentage of nuclei containing ≥ 5 FANCA foci with or without MMC treatment. n = 3 independent experiments; mean ± SEM; paired two-tailed Student’s _t_-test; ≥200 cells evaluated/experiment. d Representative immunofluorescence of FANCD2 alone and merged with DAPI (white and blue color, respectively) in MCF-10A cells expressing the indicated shRNAs (shScr, shCTDP1, or shFANCA) treated with 0.2 μM MMC for 24 h. Scale = 5 μm. e Graph representing the percentage of nuclei containing ≥ 5 FANCD2 foci with or without MMC treatment. n = 3 independent experiments; mean ± SEM; paired two-tailed Student’s _t_-test; ≥ 100 cells evaluated/experiment. f Representative immunofluorescence of γ-H2AX alone and merged with DAPI (white and blue color, respectively) in MCF-10A cells expressing the indicated shRNAs (shScr, shCTDP1, or shFANCA) treated with 0.2 μM MMC for 24 h. Scale = 5 μm. g Graph representing the percentage of nuclei containing ≥ 5 γ-H2AX foci with or without MMC treatment. n = 3 independent experiments; mean ± SEM; paired two-tailed Student’s _t_-test; ≥ 200 cells evaluated/experiment. h Analysis of FANCD2 mono-ubiquitination using cellular fractionation of MCF-10A cells (shScr and shCTDP1) with and without MMC treatment for 24 h by calculating the ratio of long (mono-ubiquitinated): short (non-ubiquitinated) proteoforms quantified by LI-COR signal intensities. (S, soluble protein fraction, including cytosol and nucleoplasm; P, pellet protein fraction, contains chromatin)
Fig. 6. CTDP1 knockdown promotes ICL sensitivity and ICL-induced apoptosis.
a Western blot confirmation of shRNA targeting CTDP1 with two independent shCTDP1 constructs (#1-TRCN0000002996 and #2-TRCN0000436164). b Representative clonogenic survival assay in MCF-10A cells expressing non-targeting shScr and shCTDP1 constructs treated with control or 5 μM melphalan. c Quantification of the clonogenic survival assays using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 5 μM melphalan (Mel). n = 3 independent experiments. Data represented as mean ± SEM; paired two-tailed Student’s _t_-test. d Clonogenic survival assay using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 0, 2, 4, 6, or 8 Gy ionizing radiation. n = 3; mean ± SEM; Asterisks indicate _p_-value ≤ 0.05; paired two-tailed Student’s _t_-test. e Clonogenic survival assay using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 0, 0.1, 0.2, 0.3, 0.4, or 0.5 μM MMC. n = 3; mean ± SEM; Asterisks indicate _p_-value ≤ 0.05; paired two-tailed Student’s _t_-test. f Clonogenic survival assay using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 0, 10, 20, 30, 40, or 50 J/m2 ultraviolet radiation. n = 3; mean ± SEM. g Clonogenic survival assay using MCF-10A cells expressing shScr or shCTDP1 #1 treated with 5 μM cisplatin or 12 μM 5-Fluorouracil (5-FU). n = 3; mean ± SEM; paired two-tailed Student’s _t_-test. h Caspase-3 activity assay using MCF-10A cells expressing shScr or shCTDP1 treated with control or melphalan for 24 h. _Y_-axis units are represented as the change in fluorescent units per microgram protein extract per hour of reaction incubation (ΔFU/μg protein/h). n = 3; mean ± SEM; paired two-tailed Student’s _t_-test. i Caspase-3 activity assay using MDA-MB-231 cells shScr or shCTDP1 treated with control or Melphalan for 0, 6, and 24 h. n = 3; mean ± SEM; paired two-tailed Student’s _t_-test. j Western blot confirmation of CTDP1 knockdown and FANCD2 expression in PD20 (FANCD2 deficient) and PD20 + FANCD2 fibroblasts. k Clonogenic survival assay using PD20 and PD20 + FANCD2 cells expressing shScr or shCTDP1 #1 treated with 0, 25, 50, 75, 100, or 200 nM MMC. n = 3; mean ± SEM. l Table of _p_-values determined using the paired two-tailed Student’s _t_-test for each listed comparison of treatment groups from results in panel k
Fig. 7. CTDP1 knockdown inhibits breast cancer cell line growth in vitro and in vivo.
In vitro growth curves of breast cell lines (a) MCF-10A, (b) T-47D, and (c) MCF-7 comparing cell proliferation between shScr and shCTDP1 expressing cells. n = 3; mean ± SEM. d Timeline and experimental design of the in vivo xenograft mouse experiments. e Western blot confirming CTDP1 knockdown in MDA-MB-231 cells used for this xenograft study. f Volumes of shScr (n = 10) and shCTDP1 (n = 12) MDA-MB-231 tumors grown in mice at Day 0, 12, 16, and 21 using Vernier caliper measurements of palpable tumors. Mean ± SEM; Asterisks indicate _p_-value ≤ 0.05; two-tailed Student’s _t_-test. g Weights of shScr and shCTDP1 MDA-MB-231 tumors obtained at necropsy. Box spans the interquartile range, horizontal line represents median, ±highest and lowest observations. h Western blot confirming CTDP1 knockdown in MCF-7 cells used for this xenograft study. i Volumes of shScr (n = 10) and shCTDP1 (n = 6) MCF-7 tumors grown in mice at Day 0, 13, and 17 using Vernier caliper measurements of palpable tumors. Mean ± SEM; Asterisks indicate _p_-value ≤ 0.05; two-tailed Student’s _t_-test. j Weights of shScr and shCTDP1 MCF-7 tumors obtained at necropsy. Box spans the interquartile range, horizontal line represents median, ±highest and lowest observations
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