p62 Promotes the Mitochondrial Localization of p53 through Its UBA Domain and Participates in Regulating the Sensitivity of Ovarian Cancer Cells to Cisplatin

Abstract

Chemotherapeutic drug-induced p53-dependent crosstalk among tumor cells affects the sensitivity of tumor cells to chemotherapeutic drugs, contributing to chemoresistance. Therefore, pharmacological targeting of p53 may contribute to overcoming drug resistance. The localization of p53 is closely related to its function. Thus, we assessed the effect of p62 on the coordination of p53 mitochondrial localization under chemotherapeutic drug treatment in ovarian cancer cells. We found that the combined use of the proteasome inhibitor epoxomicin and cisplatin led to the accumulation of p53 and sequestosome1(p62) in the mitochondria, downregulated mitochondrial DNA (mtDNA) transcription, inhibited mitochondrial functions, and ultimately promoted apoptosis by enhancing cisplatin sensitivity in ovarian cancer cells. Moreover, the ubiquitin-associated (UBA) domain of p62 was involved in regulating the mitochondrial localization of p53. Our findings suggest that the interaction between p62 and p53 may be a mechanism that determines the fate of tumor cells. In conclusion, p62 coordinated the mitochondrial localization of p53 through its UBA domain, inhibited mtDNA transcription, downregulated mitochondrial function, and promoted ovarian cancer cell death. Our study demonstrates the important role of p53 localization in tumor cell survival and apoptosis, and provides new insights into understanding the anti-tumor mechanism of targeting the ubiquitin-proteasome system in tumor cells.

Keywords: drug resistance; mitochondria; ovarian cancer; p53; p62/SQSTM1.

Figure 1

High expression of p53 is related to cisplatin sensitivity of ovarian cancer cells. (a,b) Cells were treated with cisplatin for 24 h. The cell viability of A2780 cells and SKOV3 cells were detected by MTT assay. (c) p53 protein levels were detected by western blot after treatment with cisplatin (2 μg/mL). (d,e) Cells were treated with epoxomicin(epox) for 24 h. The cell viability of A2780 cells and SKOV3 cells detected by MTT assay. (f,g) Cell viability of A2780 cells and SKOV3 cells were detected by MTT after treated with cisplatin or combined with epox (100 nM) for 24 h. # p < 0.05 vs. con, ## p < 0.01 vs. con, ### p < 0.001 vs. con., *** p < 0.001 vs. Cisplatin.

Figure 2

Epox−induced proteasome inhibition leads to mitochondrial dysfunction and UPR^mt in ovarian cancer cells. (a) A2780 cells were treated with epox (100 nM) or CDDP (2 μg/mL) or both for 24 h. Mitochondrial membrane potential detected by flow cytometry. (b) A2780 cells were treated with epox (100 nM) or CDDP (2 μg/mL) or both for 12 h. ATP production was measured based on total protein content amount. (c) A2780 cells were treated with epox (100 nM) or CDDP (2 μg/mL) or both for 12 h. Mitochondrial ROS generation was detected by flow cytometry detection. (d) A2780 cells were treated with epox (100 nM) or CDDP (2 μg/mL) or both for 12 h. Quantitative RT-PCR validation of mitochondrial DNA (mtDNA) copy number. (e) A2780 cells were treated with epox (100 nM) or CDDP (2 μg/mL) or both for 12 h. Protein levels of activating transcription factor 5(ATF5), apoptosis-related proteins in mitochondria were detected by western blot. (f) Quantitation of (B cell lymphoma-2)Bax/(B cell lymphoma-2)Bcl-2. (g,h) ATF5 nuclear protein levels detected by western blot (12 h). (i)Quantitative RT-PCR detection of mitochondrial unfolded protein response (UPR^mt) related mRNAs level. * p < 0.05 vs. con, ** p < 0.01 vs. con, *** p < 0.001 vs. con, ### p < 0.001 vs. Epox + Cisplatin.

Figure 3

Inhibition of the proteasome pathway affected the expression of respiratory chain subunits in ovarian cancer cells. (a) A2780 cells were treat with cisplatin (2 μg/mL) or epox (100 nM) or both for 3, 6, 12 h. Western blot analysis of mitochondrial subunits and mitochondrial RNA polymerase (POLRMT) levels. (b–e) A2780 cells were treat with cisplatin (2 μg/mL) or epox (100 nM) or both for 12 h. Quantitative RT-PCR detection of mtDNA-encoded respiratory chain subunits and (f) mitochondrial DNA transcription complex mRNAs level. * p < 0.05 vs. con, ** p < 0.01 vs. con, *** p < 0.001 vs. con, # p < 0.05 vs. Cisplatin, ## p < 0.01 vs. Cisplatin, ### p < 0.001 vs. Cisplatin.

Figure 4

p53 is involved in the transcription respiratory chain subunits encoded by mtDNA. Wild-type p53 were transfected into A2780 cells for 24 h and total RNA were extracted. (a) Quantitative RT-PCR detection of the transfection efficiency of wild-type p53 transfected into A2780 cells. (b–e) Quantitative RT-PCR detection of mRNAs level of respiratory chain subunits encoded by mtDNA. * p < 0.05 vs. NC, ** p < 0.01 vs. NC, *** p < 0.001 vs. NC.

Figure 5

Inhibition of proteasome pathway promoted mitochondrial localization of p53 and p62 in ovarian cancer cells. (a) A2780 cells were treat with cisplatin (2 μg/mL) or epox (100 nM) or both for 0,6,12 h. Sequestosome 1(p62) and p53 protein levels in mitochondria were measured by western blot. (b,c) A2780 cells were treat with cisplatin (2 μg/mL) or epox (100 nM) or both for 12 h. p62 and p53 protein levels in mitochondria were measured by western blot. (d,e) A2780 cells were treat with cisplatin (2 μg/mL) or epox (100 nM) or both for 12 h.p62 and p53 protein levels in nucleus were measured by western blot. *** p < 0.001 vs. con; ### p < 0.001 vs. Cisplatin.

Figure 6

Inhibition of proteasome pathway promoted mitochondrial localization of p53 and p62 in ovarian cancer cells. A2780 cells were treat with cisplatin (2 μg/mL) or epox (100 nM) or both for 12 h. (a,b) Immunofluorescence staining for p53, p62 and mitochondria in A2780 cells (bar = 30 μm).

Figure 7

Deleting the ubiquitin-associated (UBA) domain of p62 inhibited mitochondrial localization of p53 in A2780 cells. (a,b) A2780 cells were treat with cisplatin (2 μg/mL) for 12 h after transfection with different types of p62 for 24 h. Western blot analysis of p53 protein levels in the mitochondria. (c−f) A2780 cells were treat with cisplatin (2 μg/mL) for 12 h after transfection with different types of p62 for 24 h. Quantitative RT-PCR detection of mRNAs level of encoding respiratory chain subunits encoded by mtDNA. (g) A2780 cells were treated with CDDP for 24 h after transfection for 24 h. Cell viability was analyzed by MTT after cisplatin treatment for 24 h. * p < 0.05 vs. wt-p62, ** p < 0.01 vs. wt-p62, *** p < 0.001 vs. wt-p62.

Figure 8

p62 promotes the mitochondrial localization of p53 through its UBA domain and participates in regulating the sensitivity of ovarian cancer cell to cisplatin. After the combined use of epox and CDDP, p62 and p53 accumulate in the mitochondria, leading to cell death by downregulating of mitochondrial DNA transcription, UPR^mt, mitochondrial dysfunction and decreased sensitivity of A2780 cells to cisplatin, which was mediated by the UBA domain of p62. The green lightning symbol represents the use of the drug, the red text represents the detection method, the rectangle represents the experimental result, the circle represents the drug treatment, and the rhombus represents the transfection of p62 lacking the UBA domain.