CTSB Nuclear Translocation Facilitates DNA Damage and Lysosomal Stress to Promote Retinoblastoma Cell Death

Abstract

Retinoblastoma (RB) is a pernicious tumor originating from photoreceptor precursor cells that often endangers the lives of children. The purpose of our study was to further investigate the influence of cathepsin B (CTSB) nuclear translocation on RB cell death. Y79 cells were injected into the vitreous cavity of nude mice at a dose of 4 µL/mouse to establish an animal model of RB. Real-time quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, a comet assay, a Cell Counting Kit-8 (CCK-8) assay and flow cytometry were used to measure the levels of the interrelated genes and proteins and to evaluate alterations in autophagy, apoptosis, proliferation, DNA damage and cell cycle arrest. CTSB was found to be expressed at low levels in RB animal model samples and RB cell lines. Functionally, CTSB nuclear translocation promoted DNA damage, cell cycle arrest, ferroptosis and autophagy in Y79 cells and inhibited their proliferation. Downstream mechanistic studies showed that nuclear translocation of CTSB facilitates DNA damage and cell cycle arrest in RB cells by inhibiting breast cancer 1 protein (BRCA1) expression and also activates the signal transducer and activator of transcription 3/stimulator of interferon response cGAMP interactor 1 (STAT3/STING1) pathway to induce lysosomal stress, leading to ferroptosis and autophagy in Y79 cells and alleviating RB. Nuclear translocation of CTSB facilitates DNA damage and cell cycle arrest in RB cells by inhibiting BRCA1 expression and activating the STAT3/STING1 pathway and induces lysosomal stress, which eventually leads to ferroptosis and autophagy and mitigates RB.

Keywords: Autophagy; CTSB nuclear translocation; DNA damage repair; Ferroptosis; Lysosomal stress; Retinoblastoma.

Fig. 1

Expression of CTSB in retinoblastoma. A: The GSE97508 dataset was used to analyze differential gene expression. Group A: Normal group; Group B: RB group; B: RT‒qPCR was performed to measure CTSB levels; C: CTSB expression was evaluated via Western blot analysis; D‒E: CTSB expression was measured via RT‒qPCR; F‒G: CTSB protein levels were measured via Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the Control or ARPE-19 group

Fig. 2

CTSB induces autophagy-dependent death in retinoblastoma cells. A: CTSB expression was measured by Western blotting; B: Apoptosis was detected by flow cytometry; C: Cell viability was measured by a CCK-8 assay; D: Western blotting was used to measure autophagy-related protein levels. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the RB group

Fig. 3

Lysosome–nuclear translocation of CTSB promotes DNA damage. A: Nuclear translocation of CTSB was detected by immunofluorescence staining; B: DNA damage was detected by a comet assay; C: The levels of DNA damage markers were measured by Western blotting. ^***P < 0.001 vs. the RB group

Fig. 4

CTSB induces DNA damage and cell cycle arrest in retinoblastoma cells by inhibiting BRCA1. A: The expression of BRCA1 was measured by Western blotting; B: The interaction between CTSB and BRCA1 was verified by coimmunoprecipitation; C: The comet assay was used to measure DNA damage; D: The cell cycle in Y79 cells was analyzed by flow cytometry; E: Western blotting was used to measure the levels of p-BRCA1, BRCA1, CTSB and γH2AX; F: Cell cycle-related protein levels were determined by Western blot analysis. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001vs. the NC or RB group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the OE-CTSB group

Fig. 5

CTSB mediates lysosomal stress-induced ferroptosis and autophagy through the STAT3/STING1 pathway. A: The expression of STAT3 was measured by Western blotting; B: Western blotting was used to measure the levels of CTSB, p-STAT3, STAT3, and STING1; C: Ferroptosis-related protein levels were measured by Western blotting; D: Autophagy-related protein levels were measured by Western blotting; E: The content of intracellular MDA was measured by a kit; F: Flow cytometry was used to measure ROS levels; G: Apoptosis was measured by flow cytometry; H: The concentrations of inflammatory factors were measured by ELISA; I: RT‒qPCR was used to measure the expression of lysosomal markers. ^**P < 0.01, ^***P < 0.001 vs. the NC or RB group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the OE-CTSB group

Fig. 6

Activation of CTSB restrains the malignant biological behavior of retinoblastoma cells. A: The expression of Bcl-2 and Caspase-3 was measured by Western blotting; B: The levels of CTSB, p-BRCA1, BRCA1, p-STAT3, STAT3 and STING1 were measured by Western blotting; C: The levels of LC3II/I, Beclin1 and p62 were measured by Western blotting; D: Western blotting was used to measure the levels of GPX4, FTH1, SLC7A11 and HO-1; E: The levels of P18, P21 and Cyclin D1 were measured by Western blotting; F: The content of MDA was measured by a kit; G: The concentrations of inflammatory factors were assessed by ELISA. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. the RB group