PTPMT1 regulates mitochondrial death through the SLC25A6-NDUFS2 axis in pancreatic cancer cells

Abstract

Pancreatic ductal adenocarcinoma is a highly malignant cancer with poor prognosis, for which effective therapeutic strategies are urgently needed. The dual-specificity phosphatase PTPMT1 is localized in mitochondria and highly expressed in various cancers. Here, we investigated the function of PTPMT1 in pancreatic ductal adenocarcinoma. We inhibited its expression in pancreatic cancer cell lines using siRNAs or the specific PTPMT1 inhibitor alexidine dihydrochloride and observed that PTPMT1 silencing in pancreatic cancer cell lines drastically reduced cell viability, caused mitochondrial damage, and impaired mitochondrial function. Co-immunoprecipitation analysis demonstrated that PTPMT1 could interact with SLC25A6 and NDUFS2, indicating that it may modulate mitochondrial function via the SLC25A6-NDUFS2 axis. Collecively, our data highlight PTPMT1 as an important factor in pancreatic ductal adenocarcinoma and a potential therapeutic target.

Keywords: PTPMT1; Pancreatic ductal adenocarcinoma; SLC25A6; alexidine dihydrochloride.

Figure 1

PTPMT1 silencing inhibits cell proliferation and affects mitochondrial function. (A) PTPMT1 protein levels were detected in different pancreatic cancer cell lines. (B) PTPMT1 protein levels in PANC-1 cells transfected with si-PTPMT1 was analyzed using western blotting. (C) The mRNA levels of PTPMT1-silenced PANC-1 cells were analyzed by RT-qPCR. (D) PTPMT1 protein levels in MIA-PACA-II cells transfected with si-PTPMT1 were analyzed by using western blotting. (E) The mRNA levels of PTPMT1-silecned MIA-PACA-II cells were analyzed by RT-qPCR. (F) Proliferation of PANC-1 cells silenced for PTPMT1 or expressing scrambled siRNAs was assessed by CCK8 analysis. (G) Proliferation of MIA-PACA-II cells silenced for PTPMT1 or expressing scrambled siRNAs was assessed by CCK8 analysis. (H) FACS analysis of the rate of mitochondrial damage in PTPMT1-silenced and scramble-transfected PANC-1 cells. (I) Quantification of the proportion of mitochondria-damaged PANC-1 cells. (J) FACS analysis of the rate of mitochondrial damage in PTPMT1-silenced and scramble-transfected MIA-PACA-II cells. (K) Quantification of the proportion of mitochondria-damaged MIA-PACA-II cells. (L) Confocal analysis of mitochondrial morphology in PTPMT1-silenced and scramble-transfectedPANC-1 cells (Scale bar: 10 μm; Magnification: 600 ×). Data are given as mean ± SD of three independent experiments (C, E-G, I, K). ***P<0.001 (two-tailed t-test, C, E-G, I, K). β-actin (A, B, D): loading control. Data represent at least two independent experiments (A, B, D, F, G).

Figure 2

The PTPMT1 inhibitor Alexidine dihydrochloride hinders the viability of PANC-1 cells and promotes apoptosis. (A) Colony formation in PANC-1 cells treated at the indicated concentrations of Alexidine dihydrochloride (AD) (Scale bar: 500 μm; Magnification: 40 ×). (B) CCK8 analysis of the viability of PANC-1 cells incubated with AD. (C) PANC-1 cells were treated with AD for 48 hours. Analysis of protein levels via western blotting (left) and mRNA levels by RT-qPCR (right). (D) PANC-1 cells were incubated with AD for 48 hours and mitochondrial morphology was assessed via confocal microscopy (Scale bar: 10 μm; Magnification: 600 ×). (E) PANC-1 cells were treated with AD for 48 hours and mitochondrial damage was detected by FACS. (F) Quantification of mitochondrial damaged cells (E). Data are given as mean ± SD of three independent experiments (B, C, F), *P<0.05, **P<0.01, ***P<0.001 (two-tailed t-test, B, C, F). Tubulin (B): loading control. Data represent at least two independent experiments (A, C-E).

Figure 3

Identification of PTPMT1-interacting proteins via mass spectrometry. A. A total of 10,871 PTPMT1-interacting proteins were identified by mass spectrometry. B-E. Enriched terms after GO, KEGG pathway, Reactome analyses on DAVID are shown. F. A list of candidate PTPMT1-interacting proteins for further validation.

Figure 4

Identification of PTPMT1-interacting proteins by co-immunoprecipitation. (A) The interaction of PTPMT1, NDUFS2, and SLC25A6 was analyzed via immuno-precipitation using anti-Flag beads in Flag-PTPMT1-overexpressing PANC-1 cells. (B) Immunoprecipitation analysis of endogenous interaction between PTPMT1 and SLC25A6 using anti-IgG or anti-SLC25A6 antibodies. (C) Immunoprecipitation analysis of endogenous interaction between PTPMT1 and NDUFS2 using anti-IgG or anti-NDUFS2 antibodies. (D) Protein levels of NDUFS2, SLC25A6, PTPMT1, and tubulin in PTPMT1-silenced and scramble-transfected PANC-1 cells. (E) mRNA levels (RT-qPCR) of NDUFS2, SLC25A6, and PTPMT1 in PTPMT1-silenced and scramble-transfected PANC-1 cells. (F) Protein levels of the indicated proteins in SLC25A6-silenced and scramble-transfected PANC-1 cells. (G) mRNA levels (RT-qPCR) of the indicated proteins in SLC25A6-silenced and scramble-transfected PANC-1 cells. (H) Protein levels of the indicated proteins in NDUFS2-silenced and scramble-transfected PANC-1 cells. Data are given as mean ± SD of three independent experiments (E, G). ***P<0.001, two-tailed t-test (E, G). Tubulin (D, F, H): loading control. Data represent at least two independent experiments (A-D, F, H).

Figure 5

Immunohistochemistry staining and gene correlation analysis in pancreatic cancer tissues versus and adjacent non-tumor tissues. (A) Representative IHC staining of PTPMT1, SLC25A6, and NDUSF2 in PDAC and matched non-tumorous tissues. (Scale bar: 200 μm; Magnification: 40 ×) (B) IHC scores of NDUFS2 expression in PDAC and paired non-tumorous tissues. (C) IHC scores of SLC25A6 expression in PDAC and paired normal tissue. (D) IHC scores of PTPMT1 expression in PDAC and matched non-tumorous tissues. (E) Analysis of correlation between PTPMT1 and SLC25A6. (F) Analysis of correlation between PTPMT1 and NDUFS2. Data are given as mean ± SD. *P<0.05, ***P<0.001 (two-tailed t-test, B-D).