The regulation loop of MARVELD1 interacting with PARP1 in DNA damage response maintains genome stability and promotes therapy resistance of cancer cells

Abstract

The DNA damage response (DDR) plays crucial roles in cancer prevention and therapy. Poly(ADP-ribose) polymerase 1 (PARP1) mediates multiple signal transduction in the DDR as a master regulator. Uncovering the regulatory factors of PARP1 contributes to a more comprehensive view of tumorigenesis and treatment strategies. Here, we reveal that MARVELD1 acts as a mediator of DDR to perform early events and maintain genome stability. Mechanistically, PARP1 PARylates MARVELD1 at D102, D118 and D130, and in turn, MARVELD1 stabilizes PARP1 by enhancing NAA50-mediated acetylation, thus forming a positive feedback loop. MARVELD1 knockout mice and their embryo fibroblasts exhibit genomic instability and shorter half-life of PARP1. Moreover, MARVELD1 partnering with PARP1 facilitates resistance to genotoxic drugs and disrupts PARP inhibitor (PARPi) effect in PDX model of colorectal cancer (CRC). Overall, our results underline the link between MARVELD1 and PARP1 in therapeutic resistance based on DDR and provide new insights for clinical tumor therapy of PARPi.

Fig. 1. MARVELD1 regulates the genotoxic stress response of cancer cells and is associated with a poor prognosis.

a The proliferation curves of HeLa/PC and HeLa/MARVELD1 cells treated by HU, CPT or Aph for 14 days. b Immunofluorescent (IF) staining and intensity analysis of MARVELD1-V5 and γ-H2AX in HU-treated HeLa/PC and HeLa/MARVELD1 cells. c Neutral comet assays of HeLa/PC and HeLa/MARVELD1 cells treated with HU for 4 h. d The cell cycle of HeLa/PC and HeLa/MARVELD1 cells were detected at different times after HU releasement. e The endogenous MARVELD1 protein level in HeLa cells was assessed after different times or dose treatment by X-ray radiation. f The endogenous MARVELD1 mRNA level in HeLa cells was assessed after different times or dose treatment by X-ray radiation. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2. MARVELD1 interacts with DDR proteins and is a DDR mediator.

a Lysates of HU treatment HeLa/PC or HeLa/MARVELD1-Flag cells were immunoprecipitated with anti-Flag antibody. The gel pieces containing regions of interest (area shown in the bracket) were analyzed by mass spectrometry. Venn diagram showing the number of MARVELD1 interaction proteins. b The difference of protein–protein interaction (PPI) was obtained between the control and HU group. c GO analysis were performed for the MARVELD1-interacting proteins. d DDR-related GO terms and their correlated MARVELD1 interactors were constructed into a DDR-related function-protein network (the green circles mark the sensor proteins). The dot size indicates more unique peptides or a higher ratio of HU/Ctrl. e Schematic diagram show that MARVELD1 and PARP1 share interacting protein families of MCM and YWHA (right), along with their related functions (left). f MARVELD1 and PARP1 interactors were confirmed by Co-immunoprecipitation (Co-IP) and Western blotting (WB) in HeLa cells (the red squares mark the interactions after HU treatment). g MARVELD1 knockdown whether affect the interaction between PARP1 and DDR proteins was confirmed by Co-IP and WB in HeLa cells (the red squares mark the interactions).

Fig. 3. MARVELD1 binds with PARP1 and clusters in the nucleus during HU stress.

a PPI were conducted from mass spectrometry data (Fig. 2) by the common interactors of PARP1 and MARVELD1. b The interaction of MARVELD1 and PARP1 was analyzed by IP with IgG or the indicated antibody after treated with 8 mM HU (HeLa cells) or 4 mM HU (HEK293T cells) for 24 h, followed by WB. c Schematic diagram showing the domains of PARP1. ZF zinc finger, NLS nuclear localization signal, BRCT BRCA1 C-terminus, WGR Trp-Gly-Arg domain, HD helical subdomain, ART ADP-ribosyl transferase. Truncated PARP1-flag and V5-tagged MARVELD1 were co-transfected into HeLa and HEK293T cells. Lysates were analyzed by IP of Flag with a subsequent WB. d Nuclear and cytoplasmic protein extraction assays of HeLa cells with the indicated treatment for 24 h were conducted, followed by WB. e IF staining of HeLa cells was used to determine the nucleus translocation of MARVELD1 after HU treatment, and the line graphs represent the fluorescence intensity of MARVELD1 at the arrow by ZEN software. Scale bar: 10 μm. f MARVELD1 clustered in nucleus was analyzed by IF staining when HeLa cells were treated by different dose of HU, CPT or Aph. Green staining indicates MARVELD1, blue staining indicates DAPI. The relative nuclear MARVELD1 amount (integrated density/area) per cell was quantified by ImageJ and 50 cells were measured per treatment. Scale bar: 10 μm. ***p < 0.001.

Fig. 4. MARVELD1 is modified by PARP1 and clustered in the nucleus dependent on its PARylation under DDR.

a The MARVELD1 subcellular location was detected by IF staining in HeLa cells treated with olaparib or HU. Green staining indicates MARVELD1, blue staining indicates DAPI. The relative nuclear MARVELD1 amount (integrated density/area) per cell was quantified by ImageJ and 50 cells were measured per treatment. Scale bar: 10 μm. b The MARVELD1 and PARP1 protein levels were detected by WB in HeLa cells with the indicated treatments. c The PARylation of MARVELD1 was analyzed in HeLa/MARVELD1-Flag cells by IP with IgG or anti-Flag antibody, followed by WB with the PAR antibodies. d, e HeLa cells were treated with 4 mM HU or 0.5 μM CPT for the indicated times and then subjected to IP to explore the PARylation adjustment. f The MARVELD1 PARylation was analyzed in HeLa cells pretreated with 5 μM olaparib for 1 h and then treated with 4 mM HU for another 1 h. Lysates were analyzed by IP. g Nuclear and cytoplasmic protein extraction assays of HeLa/MARVELD1-WT or -3A were conducted, followed by WB. h The location change of MARVELD1 was detected by IF staining of HeLa/MARVELD1-EGFP and HeLa/MARVELD1-Flag cells after olaparib treatment. Scale bar: 10 μm. i Cell viability assays of HeLa/MARVELD1-WT or -3A to verify the cellular sensitivity in response to increasing doses of HU, CPT and Aph for 24 h. j Neutral comet assays of HeLa/PC, HeLa/MARVELD1-WT and HeLa/MARVELD1-3A cells treated with HU. Representative images and quantified tail moments are shown for each group. k Isolated nuclei of HeLa/PC, HeLa/MARVELD1-WT and HeLa/MARVELD1-3A treated with HU were subjected to MNase assays. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. MARVELD1 enhances PARP1 stability by promoting NAA50-dependent acetylation.

a PARP1 protein level in HeLa/MARVELD1 cells. b PARP1 protein level was analyzed by WB when MARVELD1-V5 and PARP1 siRNA was co-transfected in HeLa cells after 48 h. c The Ubiqutin-PARP1 was analyzed in HeLa cells. After cells treated with or without 4 mM HU for 24 h, MG-132 was added for 6 h, and lysates were subjected to IP and WB analysis. d The half-life of PARP1 was detected by WB in HeLa cells treated with CHX for the indicated time points. Relative PARP1 protein levels(PARP1/actin) were quantified and plotted in the lower panels. **p < 0.01, ***p < 0.001. e The interaction of MARVELD1, PARP1 and NAA50 was explored by Co-IP in HeLa and HEK293T cells transfected with the indicated recombinants after 48 h. f The effect of MARVELD1 on the interaction of PARP1 and NAA50 was tested by IP at 48 h after HeLa cells were transfected. g, h PARP1 protein or acetylation levels were examined in HeLa/NAA50-Flag or HEK293T/NAA50-Flag cells. The immunoprecipitated PARP1 was adjusted to equal levels to make the levels of acetylated PARP1 comparable to those of immunoprecipitated PARP1. i The Ubiqutin-PARP1 level was checked in HeLa/NAA50-Flag cells treated with or without 4 mM HU for 24 h. After HU treatment, cells were treated with MG-132 for 6 h, and lysates were subjected to IP and WB analysis. j, k PARP1 protein and acetylation level was detected in HeLa/ siNAA50 cells after 48 h transfection. l The Ubiqutin-PARP1 level was verified in HeLa/siNAA50 cells. At 48 h transfection, HeLa cells were treated with MG-132 for 6 h, and lysates were subjected to IP and WB analysis. m PARP1 protein level was analyzed in HeLa/MARVELD1 cells transfected with the NAA50 siRNA. WB was performed after 48 h transfection.

Fig. 6. MARVELD1 depletion impairs the genome stability and the levels of PARP1 protein in mice.

a Neutral comet assays of MARVELD1^+/+ and MARVELD1^−/− MEFs. Representative images and quantified tail moments are shown for each group. b γ-H2AX foci of untreated MEFs are shown. Scale bar: 20 μm. c Metaphase spreads in untreated MEFs were performed to analyze chromosome aberrations. Arrows indicate chromosome aberrations. n ≥ 100 metaphase cells in per experiment. d WB analysis of the endogenous levels of the PAR chain and PARP1 protein in MEFs (left). The quantitative data (right) of mRNA levels are from three independent experiments. e The half-life of PARP1 in MEFs was detected when cells were treated with CHX and analyzed by WB. Relative PARP1 protein levels (PARP1/actin) were quantified and plotted in the lower panels. f Cell viability assays of MEFs in response to increasing doses of HU, CPT and Aph at 48 h were performed. g Five- to eight-week-old MARVELD1^+/+ (n = 27), MARVELD1^+/− (n = 23) and MARVELD1^−/− (n = 12) littermates were subjected to 5 Gy X-ray radiation of the whole body and monitored for 30 days. h The levels of endogenous PARP1 and DNA damage were measured by immunohistochemistry (IHC) of liver and kidney tissues in mice treated as in g. Scale bar: 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7. The partnership of MARVELD1 and PARP1 induces resistance to 5-FU and olaparib in CRC.

a According to the IHC score, the patients were divided into a low MARVELD1 expression group (n = 47) and a high MARVELD1 expression group (n = 46). b The OS curves for the two groups in the CRC tissue microarray. c The correlation between PARP1 and MARVELD1 expression in the CRC tissue microarray according to the IHC score and the representative images of MARVELD1 and PARP1 protein expression in a CRC tissue microarray from 93 patients (−, +, ++, +++). d Schematic diagram showed the PDX establishment pipeline with 5-fluorouracil, olaparib or combined treatment. e The levels of MARVELD1 and PARP1 expression were analyzed by IHC in CRC PDX models. Scale bar, 100 μm. Partial magnification show the localizations of MARVELD1 and PARP1 in the nucleus. f PDX tumor sizes are shown after grouping and treatment for 3 weeks. g, h The relative tumor weight or the inhibition rates after the indicated treatments in low or high MARVELD1 expression PDX models. i The relative tumor volumes were measured each 3 days until 21 days in PDX models. *p < 0.05, **p < 0.01. j The levels of MARVELD1 and PARP1 expression were analyzed by IHC in PDX models after the indicated treatments. Scale bar, 100 μm. Partial magnification show the localizations of MARVELD1 and PARP1 in the nucleus.

Fig. 8. Model for the regulation loop of MARVELD1 interacting with PARP1.

PARP1 facilitates the nuclear translocation of MARVELD1 by PARylation to mediate the DDR early events and maintain genome stability. MARVELD1 stabilizes PARP1 by enhancing NAA50-mediated acetylation, thus forming a positive feedback loop. The partnership between MARVELD1 and PARP1 could induce therapeutic resistance.