SENP5 promotes homologous recombination-mediated DNA damage repair in colorectal cancer cells through H2AZ deSUMOylation

Abstract

Background: Neoadjuvant radiotherapy has been used as the standard treatment of colorectal cancer (CRC). However, radiotherapy resistance often results in treatment failure. To identify radioresistant genes will provide novel targets for combined treatments and prognostic markers.

Methods: Through high content screening and tissue array from CRC patients who are resistant or sensitive to radiotherapy, we identified a potent resistant gene SUMO specific peptidase 5 (SENP5). Then, the effect of SENP5 on radiosensitivity was investigated by CCK8, clone formation, comet assay, immunofluorescence and flow cytometric analysis of apoptosis and cell cycle to investigate the effect of SENP5 on radiosensitivity. SUMO-proteomic mass spectrometry combined with co-immunoprecipitation assay were used to identify the targets of SENP5. Patient-derived organoids (PDO) and xenograft (PDX) models were used to explore the possibility of clinical application.

Results: We identified SENP5 as a potent radioresistant gene through high content screening and CRC patients tissue array analysis. Patients with high SENP5 expression showed increased resistance to radiotherapy. In vitro and in vivo experiments demonstrated that SENP5 knockdown significantly increased radiosensitivity in CRC cells. SENP5 was further demonstrated essential for efficient DNA damage repair in homologous recombination (HR) dependent manner. Through SUMO mass spectrometry analysis, we characterized H2AZ as a deSUMOylation substrate of SENP5, and depicted the SUMOylation balance of H2AZ in HR repair and cancer resistance. By using PDO and PDX models, we found targeting SENP5 significantly increased the therapeutic efficacy of radiotherapy.

Conclusion: Our findings revealed novel role of SENP5 in HR mediated DNA damage repair and cancer resistance, which could be applied as potent prognostic marker and intervention target for cancer radiotherapy.

Keywords: Cancer resistance; DNA damage repair; SENP5; deSUMOylation.

Fig. 1

SENP5 was correlated radioresistance in colorectal cancer. A Expression of SENP5 in colorectal cancer tissues and normal tissues of CRC patients. These data were acquired from UALCAN (http://ualcan.path.uab.edu) B Kaplan-Meier survival analysis SENP5 high expression and low expression in Colorectal cancer (CRC) patients derived from TCGA database (log-rank p = 0.0018). These data were acquired from GEPIA2 (http://gepia2.cancer-pku.cn). C-D IHC staining and quantitative analysis of SENP5 in LARC cancer tissues with different radiosensitivity based on tumor regression grades (Radiosensitive, TRG = 0–1, Radioresistant, TRG = 3). *P < 0.05 Vs the TRG0-1 group. E Kaplan-Meier survival analysis of SENP5 in LARC patients received radiotherapy. F SENP5 mRNA expression in radiosensitive and radioresistant patients measured by RT-PCR assay. G The mRNA expressions of SENP5 in normal intestine and CRC cell lines. H Colony formation analysis of celluar radiosensitivity in CRC cells with high Vs low SENP5 expressions. ****P < 0.0001 Vs survival in SW480 cells

Fig. 2

knockdown of SENP5 increased radiosensitivity in vitro and in vivo. A Western blotting analysis of SENP5 in irradiated HCT116 and HT29 cells at 8 Gy. B-C IF staining of SENP5 at different time after irradiation (8 Gy) in HCT116 and HT29 cells. Scale bar = 10 μm. D Cell viability analysis in irradiated HCT116 cells with NC or SENP5 knockdown transfection. ****P < 0.0001, **P < 0.01 Vs NC transfected cells. E-F Colony formation analysis of radiosensitivity in HCT116 cells with NC and SENP5 knockdown cells after 0, 2, 4 and 6 Gy irradiation. ***P < 0.001 Vs NC transfected cells. G-H Cell apoptosis assay detected with a Annexin V/PI double staining method. **P < 0.01 Vs NC transfected cells. I Representative images of tumors derived from NC and SENP5 knockdown cells after local irradiation. J Growth curve of cell derived xenograft (CDX) in tumors derived from NC and SENP5 KD cells. ***P < 0.0001 Vs relative NC groups. K Immunohistochemistry staining of TUNEL and Ki67 in tumor tissues isolated from NC and SENP5 knockdown tumors after irradiation

Fig. 3

SENP5 is essential for HR-mediated DNA damage repair. A Volcano plot of differentially expressed genes in irradiated NC and SENP5 knockdown HCT116 cells (P < 0.05, |log2FC|≥1). B-C KEGG and GO enrichment of downregulated genes. D-E IF staining and quantitative analysis of γH2AX foci in NC and SENP5 knockdown HCT116 cells after 5 Gy irradiation. Scale bar = 10 μm. ***P < 0.001 Vs relative NC groups. F Representative images of comet assay in irradiated NC and SENP5 knockdown HCT116 cells. scale bar = 50 μm. G Tailed moment analysis of comet assay. ****P < 0.0001 Vs relative NC groups. H-I NHEJ and HR reporter analysis of Isel mediated DNA damage detected by flow cytometry assay. ***P < 0.001 Vs relative NC groups. J-K IF staining and quantitative analysis of Rad51 foci in NC and SENP5 knockdown cells after 5 Gy irradiation. Scale bar = 10 μm. ****P < 0.0001 Vs relative NC groups

Fig. 4

DeSUMOylation function of SENP5 is critical for its role in HR repair and radioresistance. A Schematic structure of SENP5 and the location of C713 site. B Re-expression of SENP5-wt and SENP5 C713L mutant in SENP5 knockdown cells. C-D Cell viability assay and colony formation analysis of with SENP5 knockdown HCT116 cells rescued with wild-type and C713L mutant SENP5. ****P < 0.0001, ***P < 0.001, **P < 0.01, Vs relative SENP5-wt rescued groups. E G2/M cell cycle arrest were detected with flow cytometry in SENP5 knockdown cells rescued with different mutants. Ns, non-significance, ***P < 0.001, Vs relative SENP5-wt rescued groups. F Cell apoptosis was detected in cells rescued with different mutants. Ns, non-significance, ***P < 0.001, Vs relative SENP5-wt rescued groups. G Activation of ATR related HR repair was detected by Western blotting assay in cells rescued with SENP5-wt and C713 mutant. H DNA damage repair efficacy were determined with γH2AX foci in SENP5 cells rescued with SENP5-wt and C713 mutant

Fig. 5

The protein SUMOylation landscape significantly differs between NC and SENP5 KD cells. A Flow chart of screening of deSUMOylation substrates through a SUMO-peptide pulldown assay. B Summary of differentially quantified sites and proteins (|log2FC| ≥1.5). C Subcellular localization of the identified proteins. D Top ten differential genes localized in the nucleus

Fig. 6

H2AZ is a direct deSUMOylation substrate. A-D SENP5 or H2AZ was immunoblotted in protein complex immunoprecipitated with H2AZ or SENP5 antibody in HCT116 cells (A-B) and HT29 cells (C-D), respectively. E-F Interaction of SENP5 and H2AZ were further validated in HEK-293T cells transfected with Flag-tagged SENP5 and HA-tagged H2AZ. G SUMO2/3 were detected upregulated in SENP5 knockdown cells with protein immunoprecipitated with H2AZ antibody

Fig. 7

SUMOylation of H2AZ play critical role in SENP5 mediated HR repair. A 3D structure of H2AZ was obtained from the I-TASSER server. And K120, K121 and K126 sites were observed to be responsible for DNA binding. B H2AZ-wt and H2AZ-K3R was constructed and expressed into H2AZ knockdown cells. C Activation of ATR related HR repair was detected by Western blotting assay in cells rescued with H2AZ-wt and K3R mutant. D-E Rad51 foci as key marker of HR repair was detected with IF assay in cells rescued with different mutants. Scale bar = 10 μm. ****P < 0.0001 Vs the H2AZ-wt group. F-G DNA damage were evaluated with comet assay in cells rescued with different mutants. ****P < 0.0001 Vs the H2AZ-wt group

Fig. 8

Targeting SENP5 improved the efficacy of radiotherapy in both PDO and PDX preclinical models. A Flow chart of the establishment of PDO and PDX from clinical patients. After then the PDO and PDX were exposed to indicated doses of radiation and subjected to next experiments. B Representative images of PDO transfected with lenti-virus packaged NC or SENP5 shRNA after exposed to 8 Gy irradiation. C Quantitative analysis of organoids in different groups. **P < 0.01, *P < 0.05 Vs the NC group. D Representative images of tumors isolated from PDX bearing mice in NC and SENP5 knockdown groups at the end point of observation after local irradiation. E Growth curves were generated with tumor sizes up to day 21 post-irradiation. ***P < 0.001, *P < 0.05 Vs the NC group. F Body weight of PDX tumor bearing mice were monitored every three days. G Tumor weights were measured in different groups. H-J IHC staining and quantitative analysis of γH2AX and Ki67 positive area in tumor tissues isolated in different groups. *P < 0.05 Vs the NC group. K Hypothetical model: SENP5 promotes deSUMOylation of H2AZ to promotes its removal and the recruitment of HR repair factors, which confers cancer radioresistance