DPP9 regulates NQO1 and ROS to promote resistance to chemotherapy in liver cancer cells

Chemotherapy has been the standard treatment for liver cancer. However, intrinsic or acquired drug resistance remains a major barrier to successful treatment. At present, the underlying molecular mechanisms of chemoresistance in liver cancer have not been elucidated. Dipeptidyl peptidase 9 (DPP9) is a member of the dipeptidyl peptidase IV family that has been found to be highly expressed in a variety of tumors, including liver cancer. It is unclear whether DPP9 affects chemoresistance in liver cancer. In this study, we find that DPP9 weakens the responses of liver cancer cells to chemotherapy drugs by up-regulating NQO1 and inhibiting intracellular ROS levels. In terms of mechanism, DPP9 inhibits ubiquitin-mediated degradation of NRF2 protein by binding to KEAP1, up-regulates NRF2 protein levels, promotes mRNA transcription of NQO1, and inhibits intracellular ROS levels. In addition, the NQO1 inhibitor dicoumarol can enhance the efficacy of chemotherapy drugs in liver cancer cells. Collectively, our findings suggest that inhibiting DPP9/NQO1 signaling can serve as a potential therapeutic strategy for liver cancer.


Supplemental methods
Western blot assay.Cells and tissues were lysed in RIPA lysis buffer with 1mM PMSF for 1 h on ice and centrifuged at 13,000 rpm for 30 min at 4 °C.The concentrations of total protein were measured by using BCA protein assay kit.Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes.Membranes were blocked with 3% BSA in PBS at 37 °C for 2 h and incubated with indicated antibodies overnight at 4 °C.Membranes were then washed three times with PBST followed by incubation with HRP-conjugated secondary antibodies for 1 h at 37 °C.The signals were analyzed using the ECL chemiluminescence detection system (Tanon, Shanghai, China) and Image J software was used to quantify the intensity of the bands.The experiment was independently replicated three times.
MTT assay.To determine the cytotoxicity of chemotherapeutic drugs, cells transfected with the indicated shRNA or overexpression plasmids were plated into 96-well plates at approximately 5,000 cells/well in 100 μL medium, then treated with various concentrations of chemotherapeutic drugs for 48 h.Cell viability was assessed with the MTT assay following the manufacturer's protocol.Cell survival ratio was calculated using Atreated/Acontrol  100%, where Atreated and Acontrol were the absorbance from treated and control cells after 48 h incubation, respectively.The IC50 was taken as the concentration that caused 50% inhibition of cell proliferation and was calculated by SPSS software.The experiment was independently replicated three times.
Plasmids transfection and shRNA interference.For plasmids transfection, cells were seeded in a 6-well plate and maintained in medium without antibiotics.After 24 h, the plasmids and Lipo6000 Transfection Reagent were diluted and mixed in serum-free medium according to the manufacturer's instruction.Then, the plasmids-transfection reagent complexes were added into wells.The medium was replaced after 4 h incubation and the cells were collected to perform western blot analysis after 36 h culture.For shRNA interference, DPP9, NQO1, NRF2, and KEAP1 shRNAs lentiviral particles were diluted in OptiMEM containing 6 μg/ml polybrene, and then were added to cells.After 3 days, 5 μg/ml of puromycin were used to select transfected cells.Cells transfected with the shRNAs lentiviral particles were seeded into six-well plates and western blot analysis were used to detect the protein levels of DPP9, NQO1, NRF2, and KEAP1.
RNA-sequencing.RNA-sequencing was accomplished in Personalbio.Company (Shanghai, China).In brief, total RNA of tumor tissues were extracted by Trizol Reagent (Invitrogen Life Technologies), the quality of which was measured by NanoDrop spectrophotometer (Thermo Scientific, U.S.A.).To generate sequencing libraries, the mRNA was purified by poly-T oligo-attached magnetic beads from total RNA, which was used to synthesize second strand cDNA.Then, DNA fragments were enriched by PCR reaction that were purified and quantified with Bioanalyzer 2100 system (Agilent Technologies, U.S.A.).Finally, using NovaSeq 6000 platform (Illumina, U.S.A.) to sequenced the constructed sequencing library.RNA-sequencing data generated in this study have been deposited in the Sequence Read Archive (SRA) under the accession number PRJNA1019915.
RNA extraction and qPCR assays.Total RNA was isolated using the RNA isolater Total RNA Extraction Reagent according to the manufacturer's protocol.The concentration and purity of the extracted RNA were measured with the optical densities at 260 and 280 nm.RNA samples were reverse transcribed to cDNA and subjected to quantitative PCR, which was performed with the Light-Cycler_96 RealTime PCR System (Roche) using AceQ qPCR SYBR Green Master Mix.The primer sequences used in this study were shown in Supplemental Table 2.The experiment was independently replicated three times.
CHX-chase assay.Degradation half-life of NQO1 and NRF2 was analyzed by CHXchase assay.Briefly, cells transfected with the indicated shRNA or overexpression plasmids were incubated with 25 μM of cycloheximide (CHX) to inhibit protein synthesis.Total cell lysates were collected at different times after following treatment with CHX and subjected to western blot analysis with indicated antibodies.The intensity of the bands were quantified by using Image J software.The experiment was independently replicated three times.
Co-immunoprecipitation assay.Co-immunoprecipitation assay was performed using a standard protocol.Briefly, the cell lysates were incubated with indicated antibodies overnight at 4°C, and then incubated with protein A+G agarose beads for another 4 h at 4°C.Immunoprecipitated proteins were analyzed by western blot with indicated antibodies.The experiment was independently repeated three times.
GST pull-down assay.For the GST pull-down assay, the glutathione sepharose beads conjugated with 500 ng GST or GST-DPP9-WT protein were incubated with 200 ng purified KEAP1 in GST-binding buffer at 4°C for 24 h, followed by washing 3 times for 10 minutes each with GST-washing buffer at 4°C.The samples were separated by SDS-PAGE and stained with coomassie blue.The experiment was independently replicated three times.
Measurement of intracellular ROS level.Intracellular ROS level was detected by using ROS assay kit according to the manufacturer's instructions.The fluorescence intensity was measured by using microplate reader at Ex./Em.= 488/525 nm.
Animal experiments.Female BALB/c nude mice (6 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China).All protocols for mice were approved by the Animal Ethics Committee of China Pharmaceutical University (Ethic approval number: 2022-10-019).Cells stably transfected with the indicated shRNA or overexpression plasmids were injected into subdermal space of mice (110 7 cells/per mouse).Once the tumors reached 80-100 mm 3 , the mice were treated with PBS or Cisplatin (3 mg/kg, 7 times, intraperitoneally) for 28 days.Tumor volume of mice were measured every four days.Tumor volume = (a × a × b)/2 (a, the smallest diameter; b, the largest diameter).For therapeutic experiment, cells were injected into subdermal space of mice (110 7 cells/per mouse).
Once the tumors reached 80-100 mm 3 , mice were randomly allocated into four groups, and then treated with vehicle, cisplatin (3 mg/kg, 7 times, intraperitoneally), dicoumarol (50 mg/kg, 13 times, intraperitoneally) and two drugs for 28 days.Tumor volume of mice were measured every four days.Tumor volume = (a × a × b)/2 (a, the smallest diameter; b, the largest diameter).
Immunohistochemistry assay.Immunohistochemistry (IHC) staining was performed by using immunohistochemistry kit according to the manufacturer's protocol.Briefly,

Table 2 .
The sequences of primers used in this study.